Comparison of network collaborative interaction in different immersion and different network environment.
\r\n\t
\r\n\tComputer graphics are not entirely an original topic, because it defines and solves problems using some already established techniques such as geometry, algebra, optics, and psychology. The geometry provides a framework for describing 2D and 3D space, while the algebraic methods are used for defining and evaluating equality related to the specific space. The science of optics enables the application of the model for the description of the behavior of light, while psychology provides models for visualization and color perception.
\r\n\t
\r\n\t3D computer graphics (or 3D graphics, three-dimensional computer graphics, three-dimensional graphics) is a term describing the different methods of creating and displaying three-dimensional objects by using computer graphics.
\r\n\tThe first types of graphic interpretations were put in the plane (two-dimensional 2D). Requirements for a universal interpretation led to a three-dimensional (3D) interpretation content. From these creations have arisen applied mathematics and information disciplines of graphic interpretation of content - computer graphics. It relies on the principles of Mathematics, Descriptive Geometry, Computer Science and Applied Electronics.
\r\n\t
\r\n\t3D computer graphics or three-dimensional computer graphics use a three-dimensional representation of geometric data (often in terms of the Cartesian coordinate system) that is stored on a computer for the purpose of doing the calculation and creating 2D images. The images that are made can be stored for later use (probably as animation) or can be displayed in real-time.
\r\n\t
\r\n\tObjects within the 3D computer graphics are often called 3D models. Unlike rendered (generated) images, data that are ""tied"" to the model are inside graphic files. The 3D model is a mathematical representation of a random three-dimensional object. The model can be displayed visually as a two-dimensional image through a process called 3D rendering or can be used in non-graphical computer simulations and calculations. With 3D printing, models can be presented in real physical form.
\r\n\t
\r\n\tComputer graphics have remained one of the most interesting areas of modern technology, and it is the area that progresses the fastest. It has become an integral part of both application software, and computer systems in general. Computer graphics is routinely applied in the design of many products, simulators for training, production of music videos and television commercials, in movies, in data analysis, in scientific studies, in medical procedures, and in many other fields.
Through haptic devices, users can feel partner’s force each other in collaborative applications. The sharing of touch sensation makes network collaborations more efficiently achievable tasks compared to the applications in which only audiovisual information is used. In view of collaboration support, the haptic modality can provide very useful information to collaborators.
\n\t\t\tThis chapter introduces collaborative manipulation of shared object through network. This system is designed for supporting collaborative interaction in virtual environment, so that people in different places can work on one object together concurrently through the network. Here, the haptic device is used for force-feedback to each user during the collaborative manipulation of shared object. Moreover, the object manipulation is occurred in physics-based virtual environment so the physics laws influence our collaborative manipulation algorithm. As a game-like application, users construct a virtual dollhouse together using virtual building blocks in virtual environment. While users move one shared-object (building block) to desired direction together, the haptic devices are used for applying each user’s force and direction. The basic collaboration algorithm on shared object and its system implementation are described. The performance evaluations of the implemented system are provided under several conditions. The system performance comparison with the case of non-haptic device collaboration shows the effect of haptic device on collaborative object manipulation.
\n\t\tIn recent years, there is an increasing use of Virtual Reality (VR) technology for the purpose of immersing human into Virtual Environment (VE). These are followed by the development of supporting hardware and software tools such as display and interaction hardware, physics-simulation library, for the sake of more realistic experience using more comfortable hardware.
\n\t\t\t\tOur focus of study is on real-time manipulating object by multiple users in Collaborative Virtual Environment (CVE). The object manipulation is occurred in physic-based virtual environment so the physic laws implemented in this environment influence our manipulation algorithm.
\n\t\t\t\tWe build Virtual Dollhouse as our simulation application where user will construct a dollhouse together. In this dollhouse, collaborative users can also observe physics law while constructing a dollhouse together using existing building blocks, under gravity effects. While users collaborate to move one shared-object (block) to desired direction, the shared-object is manipulated, for example using velocity calculation. This calculation is used because current available physic-law library has not been provided for collaboration. The main problem that we address is how to manipulate a same object by two users and more, which means how we combine two or more attributes of each user to get one destination. We call this approach as shared-object manipulation approach.
\n\t\t\t\tThis section presents the approach we use in study about the collaborative interaction in virtual environment so people in different places can work on one object together concurrently.
\n\t\t\tIn Collaborative Virtual Environment (CVE), multiple users can work together by interacting with the virtual objects in the VE. Several researches have been done on collaboration interaction techniques between users in CVE. (Margery, D., Arnaldi, B., Plouzeau, N. 1999) defined three levels of collaboration cases. Collaboration level 1 is where users can feel each other\'s presence in the VE, e.g. by representation of avatars such as performed by NICE Project (Johnson, A., Roussos, M., Leigh, J. 1998). Collaboration level 2 is where users can manipulate scene constraints individually. Collaboration level 3 is where users manipulate the same object together. Another classification of collaboration is by Wolff et al. (Wolff, R., Roberts, D.J., Otto, O. June 2004) where they divided collaboration on a same object into sequential and concurrent manipulations. The concurrent manipulation consists of manipulation of distinct and same object\'s attributes.
\n\t\t\t\tCollaboration on the same object has been focused by other research (Ruddle, R.A., Savage, J.C.D., Jones, D.M. Dec. 2002), where collaboration tasks are classified into symmetric and asymmetric manipulation of objects. Asymmetric manipulation is where users manipulate a virtual object by substantially different actions, while symmetric manipulation is where users should manipulate in exactly the same way for the object to react or move.
\n\t\t\tIn this research, we built an application called Virtual Dollhouse. In Virtual Dollhouse, collaboration cases are identified as two types: 1) combined inputs handling or same attribute manipulation, and 2) independent inputs handling or distinct attribute manipulation. For the first case, we use a symmetric manipulation model where the option is using common component of users\' actions in order to produce the object\'s reactions or movements. According to Wolff et al. (Wolff, R., Roberts, D.J., Otto, O. June 2004) where events traffic during object manipulations is studied, the manipulation on the same object\'s attribute generated the most events. Thus, we can focus our study on manipulation on the same object\'s attribute or manipulation where object\'s reaction depends on combined inputs from the collaborating users.
\n\t\t\t\tWe address two research issues while studying manipulation on the same object\'s attribute. Based on the research by Basdogan et al. (Basdogan, C., Ho, C., Srinivasan, M.A., Slater, M. Dec. 2000), we address the first issue in our research: the effects of using haptics on a collaborative interaction. Based on the research by Roberts et al. (Roberts, D., Wolff, R., Otto, O. 2005), we address the second issue in our research: the possibilities of collaboration between users from different environments.
\n\t\t\t\tTo address the first issue, we tested the Virtual Dollhouse application of different versions: without haptics functionality and with haptics functionality, to be compared. As suggested by Kim et al. (Kim, J., Kim, H., Tay, B.K., Muniyandi, M., Srinivasan, M.A., Jordan, J., Mortensen, J., Oliveira, M., Slater, M. 2004), we also test this comparison over the Internet, not just over LAN. To address the second issue, we test the Virtual Dollhouse application between user of non-immersive display and immersive display environments. We analyze the usefulness of immersive display environment as suggested by Otto et al. (Otto, O., Roberts, D., Wolff, R. June 2006), as they said that it holds the key for effective remote collaboration.
\n\t\t\tThe taxonomy, as shown in Figure 1, starts with a category of objects: manipulation of distinct objects and a same object. In many CVE applications (Johnson, A., Roussos, M., Leigh, J. 1998), users collaborate by manipulating the distinct objects. For manipulating the same object, sequential manipulation also exists in many CVE applications. For example, in a CVE scene, each user moves one object, and then they take turn in moving the other objects.
\n\t\t\t\tConcurrent manipulation of objects has been demonstrated in related work (Wolff, R., Roberts, D.J., Otto, O. June 2004) by moving a heavy object together. In concurrent manipulation of objects, users can manipulate in category of attributes: same attribute or distinct attributes.
\n\t\t\t\tTaxonomy of collaboration.
We construct Virtual Dollhouse application in order to demonstrate concurrent object manipulation. Concurrent manipulation is when more than one user wants to manipulate the object together, e.g. lifting a block together. The users are presented with several building blocks, a hammer, and several nails. In this application, two users have to work together to build a doll house.
\n\t\t\t\tThe scenario for the first collaboration case is when two users want to move a building block together, so that both of them need to manipulate the "position" attribute of the block, as seen in Figure 2(a). We call this case as SOSA (Same Object Same Attribute). The scenario for the second collaboration case is when one user is holding a building block (keep the "position" attribute to be constant) and the other is fixing the block to another block (set the "set fixed" or "release from gravity" attribute to be true), as seen in Figure 2(b). We call this case as SODA (Same Object Different Attribute).
\n\t\t\t\ta) Same attribute, (b) Distinct attributes in Same Object manipulation.
\n\t\t\t\t\tFigure 3 shows the demo content implementation of SOSA and SODA with blocks, hands, nail and hammer models.
\n\t\t\t\tDemo content implementation.
Even though physics-simulation library has been provided, there is no library that can handle physical collaboration. For example, we need to calculate the force of object that pushed by two hands.
\n\t\t\t\tIn our Virtual Dollhouse, user will try to lift the block and another user will also try to lift the same block and move it together to destination.
\n\t\t\t\tAfter the object reaches shared-selected or “shared-grabbed” status, the input values from two hands should be managed for the purpose of object manipulation. We created a vHand variable as a value of fixed distance between the grabbing hand and the object itself. This is useful for moving the object by following the hand’s movement.
\n\t\t\t\tWe encountered a problem of two hands that may have the same power from each of its user. For example, a user wants to move to the left, and the other wants to move to the right. Without specific management, the object manipulation may not be successful. Therefore, we decided that users can make an agreement prior to the collaboration, in order to configure (in XML), which user has the stronger hand (handPow) than the other. Therefore, the arbitration of two input values is as following (for x-coordinate movement case):
\n\t\t\t\t\n\t\t\t\t\t\tDiff = (handPos1-vHand1) - (handPos2-vHand2)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tIf abs(handPow2) > abs(handPow1)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tHand1.setPos(hand1.x-diff,hand1.y,hand1.z)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tElse if abs(handPow1) > abs(handPow2)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tHand1.setPos(hand2.x+diff,hand2.y,hand2.z)\n\t\t\t\t\t
\n\t\t\t\tAfter managing the two hand inputs, the result of the input processing is released as the manipulation result.
\n\t\t\t\tOur application supports 6DOF (Degree Of Freedom) movement: X-Y-Z and Heading-Pitch-Roll, but due to capability of our input device, we did not consider Pitch and Roll as necessary to be implemented graphically.
\n\t\t\t\t\n\t\t\t\t\t\tX-Y-Z = (handPos1-vHand1 + handPos2-vHand2)/2\n\t\t\t\t\t
\n\t\t\t\tIn Figure 4, the angle is the heading rotation (between X and Y coordinates). The tangent is calculated so that the angle in degree can be found.
\n\t\t\t\t\n\t\t\t\t\t\ttanA = (hand0.y-hand1.y)/(hand0.x-hand1.x)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\theading = atan(tanA)*180/PI\n\t\t\t\t\t
\n\t\t\t\tOrientation of object based on hands positions.
The final result of manipulation by two hands can be summarized by the new position and rotation as follows:
\n\t\t\t\t\n\t\t\t\t\t\tObject.setPos(X-Y-Z)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tObject.setRot(initOri.x+heading, initOri.y, initOri.z)\n\t\t\t\t\t
\n\t\t\t\tBased on two user manipulation, three users manipulation can be calculated easily following the same algorithm. We have to choose which two hands against the other one hand (see Figure 5) based on hand velocity checking.
\n\t\t\t\tExample of three users manipulation, Hand 0 and Hand 1 against Hand 2.
After calculation, manipulation that made when three hands want to move an object together can be found below.
\n\t\t\t\t\n\t\t\t\t\t\tFor each x, y, and z direction, check:\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tIf abs(vel_hand0) >= abs(vel_hand1 + vel_hand 2)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tHand1 and hand2 follow hand0\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tElse if abs(vel_hand1) >= abs(vel_hand0 + vel_hand 2)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tHand0 and hand2 follow hand1\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tElse if abs(vel_hand2) >= abs(vel_hand0 + vel_hand 1)\n\t\t\t\t\t
\n\t\t\t\t\t\n\t\t\t\t\t\tHand0 and hand1 follow hand2\n\t\t\t\t\t
\n\t\t\t\t\n\t\t\t\t\t(1) Virtual Dollhouse\n\t\t\t\t
\n\t\t\t\tWe have built Virtual Dollhouse as our CVE. Our Virtual Dollhouse application is made based on OpenGL Performer (Silicon Graphics Inc. 2005) and programmed in C/C++ language in Microsoft Windows environment. VRPN server (Taylor, R. M., Hudson, T. C., Seeger, A., Weber, H., Juliano, J., Helser, A.T. 2001) is used to provide management of networked joysticks to work with the VR application. We use NAVER Library (Park, C., Ko, H.D., Kim, T. 2003), a middleware used for managing several VR tasks such as device and network connections, events management, specific modeling, shared state management, etc.
\n\t\t\t\tThe physics engine in our implementation is an adaptation of AGEIA PhysX SDK (AGEIA: AGEIA PhysX SDK) to work with SGI OpenGL Performer\'s space and coordinate systems. This physics engine has a shared-state management so that two or more collaborating computers can have identical physics simulation states. Using this physics engine, object\'s velocity during interaction can be captured to be sent as force-feedbacks to the hands that are grabbing the objects.
\n\t\t\t\tThe architecture of our implementation can be seen in Figure 7.
\n\t\t\t\tVirtual Dollhouse as VCE.
System architecture of the implementation.
To enable easy XML configuration, the application is implemented in a modular way into separate DLL (Windows\' dynamic library) files. Using pfvViewer, a module loader from SGI OpenGL Performer, the dynamic libraries are executed to work together into one single VR application. All configurations of the modules are written in an XML file (with.pfv extension). The modules can accept parameters from what are written in the XML file, such as described in this figure below.
\n\t\t\t\tConfiguration of physics simulation in XML file.
\n\t\t\t\t\t(2) Three-Users Design and Implementation\n\t\t\t\t
\n\t\t\t\tInteraction status on the same object by three users is shared by showing several different states. These states are touched and selected by one, two, or three users. For user’s graphical feedback purpose, these states are described by color yellow, cyan, green, magenta, red, and blue respectively (Figure 9).
\n\t\t\t\tGraphical feedback for three-users.
Each user is represented by one hand avatar. We modify our previous algorithm in order to check all these “touch” and “select” status easier. We check object status instead of hand status that we used in our previous algorithm. “Select” status only can happen after “touch” status. In a frame, we will check the touching status for each object and define how many hand and which hand that touches the object. Still in the same looping of each object, we check the selecting status of that object and doing manipulation for that object based on how many hand selects that object.
\n\t\t\t\tWe made our application fit with Joystick and SPIDAR (Sato, M. 2002) - WAND input. These devices will be used in our testing to give input to our simulation. BUTTON_PRESSED in the figure below represents the “selecting or grabbing” button from Joystick or WAND button.
\n\t\t\t\tAlgorithm of the object and hand status.
The algorithm for shared-object manipulation is extended from two user manipulation into three user manipulation. The calculation of movement for three users is made based on two users’s manipulation. The different is we have to choose which two hands against the other one hand based on hand velocity checking.
\n\t\t\tAs result of our approach, we present the comparative study of two users and the simulation result of three users.
\n\t\t\t\tWe have done comparative study for two users. Two users manipulate the same object together concurrently in: 1) PC and PC environment through LAN inside KIST and the Internet between KIST,Korea and Oita University, Japan through APII-Hyunhae-Genkai Network, 2) CAVE (Cruz-Neira, C., Sandin, D. J., DeFanti, T. A., Kenyon, R.V., Hart, J.C. 1992) and PC environment through LAN. The test also includes the comparative study between haptic (with force feedback) and non-haptic (no force feedback) device. We will use joystick as input device for PC environment. In the CAVE system, the input devices that used are SPIDAR for movement and WAND for object selecting/grabbing button.
\n\t\t\t\t\n\t\t\t\t\tTable 1 shows our experiment result. We test five times and calculate average time for completing the collaborative interaction.
\n\t\t\t\tNetwork Collaborative Interaction (NCI) Comparative Study.
\n\t\t\t\t\t\t\t | PC-PC | \n\t\t\t\t\t\t\tPC-PC | \n\t\t\t\t\t\t\tCAVE-PC | \n\t\t\t\t\t\t
\n\t\t\t\t\t\t\t | Non Force-Feedback | \n\t\t\t\t\t\t\tForce-Feedback | \n\t\t\t\t\t\t\tForce-Feedback | \n\t\t\t\t\t\t
LAN I nside KIST | \n\t\t\t\t\t\t\t29.096 s | \n\t\t\t\t\t\t\t21.344 s | \n\t\t\t\t\t\t\t16.676 s | \n\t\t\t\t\t\t
Internet (bw Korea and Japan) | \n\t\t\t\t\t\t\t43.55 s | \n\t\t\t\t\t\t\t36.92 s | \n\t\t\t\t\t\t\t- | \n\t\t\t\t\t\t
Comparison of network collaborative interaction in different immersion and different network environment.
We have implemented an application for CVE based on VR systems and simulation of physics law. The system allows reconfiguration of the simulation elements so that users can see the effects of the different configurations. The network support enables users from different places to work together when interacting with the simulation, and see each other\'s simulation results.
\n\t\t\tFrom our series of testing of the application over different networks and environments, we can conclude that the use of haptics functionality (force-feedback device) is useful for users to feel each other\'s presence. It also helps collaboration to be performed more effectively (no time wasted). However, network delays caused problems on the haptics smoothness. In the future, we will update our algorithm by studying the possible solutions like indicated by Glencross et al. (Glencross, M., Jay, C., Feasel, J., Kohli, L., Whitton, M. 2007).
\n\t\t\tWe also conclude that the use of tracker-type input device like SPIDAR is more intuitive for a task where users are faced with a set of objects to select and manipulate. From the display view of point, immersive display environment is more suitable for simulation of dealing with object manipulation that requires force and weight feeling, compared to non-immersive display environment such as PC.
\n\t\tThis work was supported in part by KIST (Korea Institute of Science & Technology) through Development of Tangible Web Technology Project.
\n\t\tThe skin pigmentation is formed by the synthesis of melanin in the melanocytes. Melanocyte is a kind of epithelial cells mainly locating basal cell layers of epidermis, and a few number of melanocytes are located in mucosa. Pigment granules constituted with melanin can distribute and transport to neighboring keratinocytes [1]. Mutations in melanocortin-1-receptor (MC1R) are pivotal for human skin’s tanning and pigmentation. MC1R belongs to a G-protein-coupled receptor (GPCR) that is expressed in epidermal melanocytes in a preferential manner [2]. α-Melanocyte-stimulating hormone (α-MSH), the GPCR’s ligand, is a propigmentation hormone which is generated and secreted by both keratinocytes and melanocytes in the skin. After UV irradiation, α-MSH can activate GPCR. Pro-opiomelanocortin (POMC) is a multicomponent precursor for α-MSH (melanotropic), ACTH (adrenocorticotropic), and the opioid peptide β-endorphin, and α-MSH and other bioactive peptides are the cleavage products of POMC [2]. Normal synthesis of α-MSH and ACTH is extremely important to constitutive human pigmentation and the cutaneous response to UV [2].
\nIn melanocytes, the amount and type of pigment production are regulated by MC1R. So MC1R is an important determiner of skin phototype, sensitivity to UV radiation-induced damage, and skin cancer risk [3]. The heterotrimeric G proteins consist of α, β, and γ subunits. Upon ligand binding, a signal is transmitted by GPCRs to heterotrimeric G proteins, which results in the separation of the α subunit from the Gβγ subunit of G proteins. ATP is catalyzed to be directly transformed to cAMP by the G proteins of the Gαs class and cAMP is in charge of melanogenesis including the sensitization of tyrosinase in melanin biosynthesis upon being activated by ligands such as α-MSH [4].
\np53 is not only a transcriptional factor but also a tumor-suppressor protein, which is documented to directly sensitize the transcription of a lot of genes including those that control cell cycle, apoptosis, and others. POMC/MSH inducement by UV irradiation in skin is directly regulated by p53 and POMC promoter is stimulated in response to UV irradiation. p53 involves in UV-independent pathologic pigmentation and could imitate the tanning response [1]. Dipyrimidine C to T substitutions including CC to TT frameshift mutations in the p53 gene can be uniquely induced by UV in the skin of UV-irradiated mice months before tumor development [5]. In addition, p53 has been demonstrated to be necessary to the presentation of “sunburn cells,” which are a sign of sunburns [5].
\nDUH characterized by extensively mottled pigmentation is a heterogeneous disorder, which was first diagnosed by two Japanese researchers in two generations of two pedigrees for about 80 years [6, 7]. Similar Chinese DUH pedigrees with dyschromatosis symmetrica hereditaria (DSH) with autosomal dominant DUH had been reported by us in 2003 [8] and diagnosed as DUH rather than DSH afterward. Although novel mutations of SASH1 have been identified to be associated with dyschromatosis universalis hereditaria [9], less pathogenesis of DUH has not been investigated. The pathogenesis of DUH remain unclear and indefinite for 80 years [7].
\nSASH1, a previously described novel family of putative adapter and scaffold proteins transmitting signals from the ligand to the receptor, was first showed to be a candidate tumor-suppressor gene in breast cancer and colon carcinoma [10, 11, 12]. Our previous study demonstrates that SASH1 interacts with Gαs, the downstream player of α-MSH/MC1R signaling pathway [13]. Our previous report indicated that in the several affected DUH individuals, hyperpigmented macules became more obvious after strong UV irradiation particularly in summer [8], but no further investigations was performed to identify the reasons of photosensitivity [14]. The significance of expression of p53/POMC/α-MSH in UV-photopigmentation response and UV-independent hyperpigmentation has been explained [1]. However, few investigations were performed to reveal that the mutations in SASH1 gene are related to hyperpigmentation and how these mutations result in hyperpigmentation phenotype.
\nIn a word, we assume that a novel p53/POMC/α-MSH/Gαs that SASH1 involves in regulating UV-photopigmentation response and pathological hyperpigmentation phenotype.
\nTwo Chinese pedigrees recruited from the Henan and Yunnan provinces of China and one American pedigree with typical features of DUH were involved in this study. Three DUH pedigrees with an autosomal dominant inheritance pattern were diagnosed by skilled clinical dermatologists. The small American pedigree only offered peripheral blood samples of the affected individuals for investigations. This study was recognized by the ethical review committees from the appropriate institutions. Genotyping was implemented, and the two-point LOD score was counted as our previous description [8]. In total, 50 family members and 500 normal individuals (controls) involved in the research were provided with informed consent and specimens of peripheral blood DNA were acquired from all obtainable family members. PCR and sequencing were executed as our previous description [8]. ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, CA) was used to perform the sequencing on an ABI PRISM 3130 DNA Analyzer (Applied Biosystems) and sequence analysis software, version 3.4.1 (Applied Biosystems) were used to analyze the data. Sequencher 4.10.1 (Gene Codes Corp.) was used to compare the sequence data with SASH1 reference sequence (GenBank NM_015278.30). Nucleotide numbering reflects complementary DNA (cDNA) numbering, with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence [8].
\nWild-type and mutant SASH1-PEGFP-C3 and wild-type and mutant SASH1-PBABE-Flag-puro were constructed according to the protocol of our previous study [13]. To generate the p53-HA-Pcna3.0, POMC-myc-Pcdna3.0 and Gαs-Pegfp-C3 vectors, high fidelity DNA polymerase (Phusion Hot Start High Fidelity Polymerase from New England Biolabs, Inc. or GXL Polymerase from Takara) and the primers indicated in Table 1 were used to amplify the bacteria (obtained from Han Jiahuai Lab, Xiamen University, Xiamen, China) containing the vector of full-length CDS sequences of and Gαs, p53 and POMC. Mammalian expression vector (Invitrogen) via the relative restriction sites and sequenced.
\nName of primers or probes | \nSequences (5′–3′) | \n
---|---|
Gαs forward primer (SalI site included) | \nACGCGTCGACATGGGCTGCCTCGGGAAC | \n
Gαs reverse primer (XhoI site included) | \nCCGCTCGAGTTAGAGCAGCTCGTACTGACG | \n
p53 forward primer (BamHI site included) | \nCGCGGATCCGCCACCACCATGGAGGAGCCGCAGTCAGATCCTA | \n
p53 reverse primer (XhoI site included) | \nCCGCT CGAG TCAGTCTGAG TCAGGCCCTTCTGT | \n
POMC forward primer (BamHI site included) | \nCGCGGATCCATGCCGAGATCGTGCTGC | \n
POMC reverse primer (XhoI site included) | \nCCCAAGCTTTCACTCGCCCTTCTTGTAGGCGTTCTTGAT | \n
SASH1 probe 1# | \nGCCCAAGCTTTCACACTTGTTT | \n
SASH1 probe 2# | \nCCAAGACTTGCTAGAAGGAACGAGTCG | \n
SASH1 probe 3# | \nCGTGGCCACCTAG ACCCGAGGTG | \n
Sequences of primers or probes used in gene cloning and EMSA in this study.
SK-MEL-28, HEK-293T, and A375 cells were cultured according to our previous description [15]. Normal human epithelial melanocytes (NHEMs, C-12402, PromoCell, Germany) were maintained in M2 medium. Lipofectamine 2000 (11668-027, Invitrogen) as previously described [15, 16] or Entranster-D (18668-01, Engreen Biosystem Co., Ltd.) or polyethyleneimine (PEI) prepared by ourselves were, respectively, used for the transfection of SK-MEL-28, A375, B16, and HEK-293T cells. The transfected A375 and SK-MEL-28 cells were screened with 1.5 μg/ml puromycin or 2.0 μg/ml G418 to construct stable cell lines. Wild-type and mutant SASH1-pEGFP-C3 or co-transfected with wild-type SASH1-Pbabe-Flag-puro and Gαs-Pegfp-C3 vectors were transiently introduced into HEK-293T cells for immunoprecipitation experiments. p53-HA-Pcdna3.0, POMC-myc-Pcdna3.0, Gαs-Pegfp-C3, and wild-type SASH1-pEGFP-C3 according to pairwise combination were introduced into NHEMs and HEK-293 or HEK-293T cells to detect the expression of exogenous p53, POMC, Gαs, and SASH1 using PEI or PromoFectin (PK-CT-2000-MAC-1, PromoCell).
\nGαs-GFP, HA-p53, myc-POMC, and GFP-SASH1 recombined vector were introduced into HEK-293T cells. 24 h later, Entranster™-R transfection reagent (18668-06, Engreen Biosystem Co., Ltd) was used to transfect Gαs- and POMC-specific siRNAs synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) to silence the expression of exogenous Gαs, p53, and SASH1 in the transfected HEK-293T cells. The sense/antisense sequences of each siRNA for Gαs, POMC are documented in Table 2.
\nGene name | \nForward primer (5′–3′) | \nReverse primer (5′–3′) | \n
---|---|---|
SASH1 | \nCGGGAAACGTCAAGTCGGA | \nATCTCCTTTCTTGAGCTTGAG | \n
TYRP1 | \nCACAGGCACAGGTACCACCTC | \nCTGAACTACCCTAGGTCTTCGTT | \n
Pmel17 | \nAAGGTCCAGATGCCAGCTCAA | \nCTTTCACGGCTCTAGGACGTC | \n
Rab 27a | \nAACTAGTGCTGCCAATGGGACA | \nTTTGATCGCACCACTCCTTC | \n
Gαs | \nGTCCTTGCTGGGAAATCG | \nCGCAGGTGAAATGAGGGTAG | \n
p53 | \nCCACCATCCACTACAACTACAT | \nTCCCAGCACAGGCACAAA | \n
POMC | \nAGTTCAAGAGGGAGCTGACTGG | \nCATGAAACCGCCGTAGCG | \n
GAPDH | \nCACCCACTCCTCCACC TTTG | \nACCACCCTGTTGCT GTAGCC | \n
Gαs siRNA 1 | \nGAGGACUACUUUCCAGAAUTT | \nAUUCUGGAAAGUAGUCCUCTT | \n
Gαs siRNA 2 | \nGCAGCUACAACAUGGUCAUTT | \nAUGACCAUGUUGUAGCUGCTT | \n
POMC siRNA1 | \nACCUCACCACGGAAAGCAATT | \nUUGCUUUCCGUGGUGAGGUTT | \n
POMC siRNA2 | \nAGUACGUCAUGGGCCACUUTT | \nAAGUGGCCCAUGACGUACUTT | \n
GAPDH | \nGUAUGACAACAGCCUCAAGTT | \nCUUGAGG CUGUUGUCAUACTT | \n
Negative control | \nUUCUUCGAACGUGUCACGUTT | \nACGUGACACGUUCGG AGAATT | \n
Primers used for site directed mutagenesis, real time RT-PCR and RNAi.
Procedure of the pull-down assay, LC-MS/MS analyses, database search, and bioinformatic analyses for functional classification are mainly as performed as our previous description [13].
\nTransfected HEK-293T cells or HEK-293 cells or NHEMs with ectopic exogenous genes were washed in a gentle way for three times with PBS and lysed in IP-western blot lysis buffer (P0013, Beyond Time BioScience and Technology Company) in the presence of a complete protease inhibitor cocktail, 1 μM sodium orthovanadate, and 1 mM sodium fluoride per 10 cm dish on ice for 20 min to acquire lysisprotein. Cell lysates were centrifuged for 10 min at 12,000 rpm at 4°C. 600 μl of supernatants of cell lysates were pre-cleaned with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) for 1 h. GFP-Tag (7G9) mouse mAb (Shanghai Abmart, Inc.) or DYKDDDDK-Flag-Tag mouse mAb (Shanghai Abmart) or HA-Tag mouse mAb (Shanghai Genomics) was used to immunoprecipitate the corresponding proteins at 4°C for 2 h. Then, the cell lysates were mixed with 20 μl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) at 4°C for 10 h for co-immunoprecipitation or immunoprecipitation analyses. Finally, immunoprecipitates were washed for three times with PBS and subjected to western blotting. GFP-Tag mouse Ab, Flag-tag mouse mAb, DYKDDDDK-Flag mouse mAb, GAPDH mouse mAb and anti-β-tubulin mouse mAb (Shanghai Abmart, Inc.), anti-Gαs rabbit polyclonal Ab (Gene Tex, Inc.), myc-tag mAb and HA-tag mouse mAb (Shanghai Genomics), SASH1 Rabbit mAb (Bethyl Laboratories, Inc.), TYRP1 (TA99) mouse mAb and melanoma gp100 Rabbit mAb (Abcam), Rab 27a mouse mAb (Abnova) were used for immunoblotting assay as previously described [17, 18].
\nAll participating patients in this study were given the written informed consent regarding tissue and data use for scientific purposes. Epithelial tissues from affected individuals with the Y551D SASH1 mutation from pedigree I were fixed and embedded in paraffin. Paraffin sections (5 μm) were baked at 56°C for 3 h, and then deparaffinized and rehydrated using xylene and an ethanol gradient. SASH1 monoclonal antibody, rabbit anti-ACTH antibody, MiTF polyclonal antibody, the antibodies of melanogenesis related molecules including HMB45, TYRP1, and Rab 27a and p53 monoclonal antibody was used to bind the corresponding proteins on paraffin sections, respectively. Finally, counterstaining of hematoxylin was performed and the sections were photographed under the positive position microscope BX51.
\nA375 stable cells with ectopic wild-type or mutant SASH1 in 6-well chamber slides were analyzed with indirect immunofluorescence analysis. SASH1 rabbit mAb (Betheyl Laboratories, Inc.) and DYKDDDDK-Flag mouse mAb (Shanghai Genomics, China) were used to assess SASH1 localization and expression, as described previously [13].
\nThe melanin staining of paraffin sections obtained from epithelial tissues were performed as our previous descriptions and observed under a light microscope [18].
\nTRIzol reagent (Invitrogen) was used to isolate the total RNA from the different groups of SK-MEL-28 cells. Reverse transcription was performed according to the manufacturer’s protocol for the PrimeScript™ RT Reagent Kit (DRR037A, TaKaRa) for qRT-PCR. The sense and antisense primer sequences for SASH1, TYRP1, Pmel17, Rab27a, Gαs, POMC, and GAPDH are showed in Table 2. The PCR products were identified by agarose gel electrophoresis. The Applied Biosystems 7500 System was applied to detect the expression of corresponding genes with SYBR Premix Ex Taq™ (DRR041A, TaKaRa). The quantity of each mRNA was normalized to that of GAPDH mRNA.
\nHuman foreskin tissues from a 14 year-old boy were irradiated for enough time under a ultraviolet phototherapy instrument (NBUVB SS-05, Sigma) to reach the expected UV intensity. The irradiated tissues were fixed in 10% formalin and embedded in paraffin for immunohistochemistry analyses. We conformed to the guidelines of the World Medical Assembly (Declaration of Helsinki) to acquire the human foreskin tissues. In vitro UV experiments were mainly referred to the protocol of our institute [19]. HEK-293T cells and NHEMs transiently with ectopic myc-POMC were subcultured to approximately 70–80% confluence and were irradiated with 100 mJ/cm2 UVC delivered via a HL-2000 HybriLinker with a 254 nm wavelength and followed by the indicated recovery time. Finally, immunobloting was performed to identify the corresponding proteins’ levels.
\nThree probes binding with/without biotin targeting the promoter sequence of SASH1 gene were synthesized. The sequences of probes were as indicated in Table 1. Electrophoretic mobility shift assay was performed as described as the protocol provided with LightShift® Chemiluminescent EMSA (20148, Thermo Scientific, Pierce Biotechnology) to detect the bindings of SASH1 with p53 [18].
\nThe data are indicated as mean ± standard error of the mean (SEM)s. The homogeneity of variance test was first used to analyze the variance homogeneity of data and the data were analyzed the change of variable test. Statistical significance was determined by a one-factor analysis of variance (ANOVA) using LSD on SPSS version 16.0 to produce the required p-values. Cartograms were plotted with GraphPad Prism 5.
\nThe gene that is responsible for DUH had been localized to chromosome 6q24.2-q25.2. The 10.2 Mb region on chromosome 6 (6q24.2-q25.2) containing more than 50 candidate genes is flanked by the markers D6S1703 and D6S1708 [8]. Direct sequencing of the PCR products of exons amplified from genomic DNA of affected, unaffected, and control individuals was performed to screen the selected genes in this region for possible pathological mutations. 50 candidate genes were sequenced. Finally, in the probands in each of the two nonconsanguineous Chinese DUH-affected pedigrees (families I and II) and in one nonconsanguineous American DUH-affected pedigree (family III), three heterozygous mutations encoding amino acid substitutions in SAM and SH3 domain containing I (SASH1) were found in the three pedigrees. The substitution mutations in SASH1 gene were as follows: a TAC → GAC substitution at nucleotide 2126 in exon 14, causing a Y551D (p.Tyr 551 Asp) mutation at codon 551 in family I, a CTC → CCC substitution at nucleotide 2019 in exon 13, causing a L515P (p.Leu to Pro) mutation at codon 515 in family II, and a GAA → AAA substitution at nucleotide 2000 in exon 13, resulting in a E509K (p.Glu to Lys) mutation in family III. These sequence alterations were identified in all of the affected pedigree members, but were not observed in unaffected pedigree members, correlating the presence of the mutations with the presence of the phenotype. In any of the 500 normal controls or in any of the current databases, including the HapMap database, these three SASH1 mutations were not found [18]. So, these three mutations are impossible to be common single nucleotide polymorphisms (SNPs) [8].
\nIn A375 stable cells with ectopic SASH1 gene mutants, mutated SASH1 were found to be significantly up-regulated (Figure 1B). Western blot showed that up-regulation of SASH1 was found in A375 cells stably expressing either wild-type (WT-A375 cells) or mutant SASH1, when compared to the expression of endogenous SASH1 in A375 cells with expression of pBABE-puro empty vector (VECTOR-A375 cells) or BLANK-A375 cells (Figure 1B) [18].
\nIncreased SASH1 expression is induced by mutations in SASH1 in vitro and in vivo. (A) Substitution mutation sites in the SASH1 gene in three DUH pedigrees. (B) Differential and increased expression of mutant SASH1 proteins is detected compared to that of wild-type SASH1 in different A375 cells by immunoblot. (C) Immunohistochemistry detection of SASH1 and Mitf. Heterogeneous SASH1 protein was detected in all of the DUH-affected epithelial layers compared to that of normal controls (NC). Heterogeneous distribution of melanocytes is detected in the epithelial layers of DUH-affected individuals using Mitf compared to that of normal controls. 400× magnification. Scale bar = 20 μm. The representative positive cells of SASH1 and Mitf were denoted by red arrows.
To identify the stability of SASH1 proteins, 20 μg/ml of the protein synthesis inhibitor cycloheximide (CHX) was used to treat HEK-293T stable cells with ectopic wild-type or mutant SASH1 expressing for the indicated times to assess the half-life of SASH1. SASH1 protein levels were induced to decrease by CHX treatment in a time-course-dependent manner. Wild-type SASH1 levels was decreased with a half-life of approximately 4 h, however, mutant SASH1 proteins began to degrade with CHX treatment for 6 h or longer. Therefore, it is deduced that the three mutant SASH1 proteins were more steady than the wild-type, confirming the conclusion offered above that expression levels of mutated SASH1(s) are higher levels than that of wild-type (Figure 2A and B). Endogenous SASH1 was not a stable protein with an half-life of approximately 3 h as identified by western blot (Figure 2C) [18].
\nEndogenous SASH1 protein is unstable and mutation of SASH1 induces the heterogeneous expression of SASH1 in vitro. (A) Mutant SASH1 proteins are more stable than the wild-type SASH1 protein. Stable HEK-293T cells were treated with CHX (20 μg/ml) for the indicated times and analyzed by western blotting. The amount of SASH was quantified by densitometry and normalized to β-tubulin. CHX resulted in the degradation of wild-type SASH1 protein, which had a half-life of 4 h. Under a 6-h or longer treatment with CHX, CHX began to induce the degradation of mutant SASH1 proteins. (B) The intensity of GFP-SASH1 was quantified by densitometry and normalized to β-tubulin (n = 3). (C) Endogenous SASH1 is an unstable protein. HEK-293T cells were deprived of FBS for the indicated time and lysed and subjected to western blot to detect the endogenous SASH1 levels.
The subcellular localization of SASH1 in A375 cells and skin epithelial layers was further characterized. A homogenous pattern of expression of SASH1 protein was observed in VECTOR-A375 cells and the skin epithelial layers from normal controls (Figures 1C and 3-a4). However, heterogeneity expression of SASH1 protein was showed in WT-A375 cells and mutant-A375 cells (Figure 3-b4–e4) as well as in the epithelial tissues of affected individuals (Figure 1C). In addition, as identified by Mitf, a melanocyte indicator, most of the SASH1-positive cells were Mitf-nucleic positive-melanocytes in the epithelial tissues of DUH-affected individuals. These Mitf-nucleic positive-melanocytes in the affected epithelial layer showed a heterogeneous distribution compared to those of unaffected individuals (Figure 1C). Some Mitf-tenuigenin-positive staining is of false positivity (Figure 1C). Melanocytes or SASH1-positive epithelial cells not only localized at the basal layers, but also the suprabasal layers of the affected epidermal tissue, the phenomenon of which coincides with our previous descriptions that SASH1 mutations promotes melanocyte movement from the affected basal layers to the superficial ones [13].
\nSubcellular localization of SASH1. The fluorescence signals that were detected by confocal microscopy indicate that the overexpression or mutation of SASH1 results in the heterogeneous expression of SASH1 in vitro in A375 stable cells. The green fluorescence represents the Flag-tag label. Both exogenous and endogenous SASH1 are labeled with a red fluorescent tag. The nuclei are labeled with DAPI (in blue). The yellow fluorescence indicates the overlap of the green and red fluorescent staining. The red arrowheads indicate the activated SASH1-Flag fusion proteins that were expressed in the cytoplasm of WT-A375 cells or mutant-A375 cells. The blue arrowheads indicate the regions that do not express the activated SASH1-Flag fusion protein in the cytoplasm of WT-A375 or mutant-A375 cells. The endogenous SASH1 presents a uniform pattern of expression in all of the VECTOR-A375 cells (Figure 3A-a4). Scale bar = 5 μm.
SASH1 contains two functional domains, SAM and SH3 domain, which indicates SASH1 may plays an important role in a signaling pathway acting as a signaling molecule adapter or an associated scaffolding protein [8, 9]. Therefore, we performed a pull-down assay and a mass spectrometry analysis to investigate which signaling pathways are regulated by SASH1. Pull-down experiments and nanoflow LC-MS/MS analysis demonstrated that SASH1 interacts with Gαs and CALM, both of which are important in melanogenesis process (Tables 3 and 4) in WT-A375 cells. Gαs connects receptor-ligand associations with the activation of adenylyl cyclase and many cellular responses, serving as a pivotal player in the conventional signal cascades [20]. To investigate the associations between SASH1 and Gαs, HEK-293T cells were co-transfected with Flag-SASH1 and GFP-Gαs. Exogenous SASH1 was immunoprecipitated with both exogenous Gαs (GFP-Gαs) and endogenous Gαs. Immunoprecipitates of exogenous SASH1 had different observed band sizes of Gαs (Figure 4B and C) [18].
\nProtein name | \nScore | \nProtein possibility | \nTotal peptide | \nUnique peptide | \n
---|---|---|---|---|
SASH1 | \n200.3 | \n1 | \n37 | \n20 | \n
Gαs | \n20.2 | \n1 | \n8 | \n5 | \n
CALM1 | \n10.2 | \n1 | \n7 | \n3 | \n
Proteins interacting with SASH1 were identified by MS analysis.
Affinity-purified proteins were identified by MS analysis and the detailed peptide sequences are summarized in Table 2.
Protein name | \nPeptide sequence | \n
---|---|
SASH1 (O94885) | \nK.KPSTEGGEEHVFENSPVLDERS R.AVLLTAVELLQEYDSNSDQSGSQEKL K.GEDVGYVASEITMSDEERI R.VSQDLEVEKPDASPTSLQLR.S R.VHTDFTPSPYDTDSLKI K.LLEEEDLDELNIRD K.LHAEGIDLTEEPYSDKH K.PGAGTSEAFSR.L KPLFFDGSPEKPPEDDSDSLTTSPSSSSLDTWGAG K.MGTFFSYPEEEKA KMITIEEALARL RSLHVGSNNSDPMGKE SLHVGSNNSDPMGK ITIEEALAR MITIEEALARL RGVDLETLTENKL IPSQPPPVPAK TIEEALAR KYFWQNFRK SALYSGVHK | \n
Gαs (P63092) | \nEAIETIVAAMSNLVPPVELANPENQFR YTTPEDATPEPGEDPR IEDYFPEFAR MFDVGGQR VLTSGIFETK | \n
CALM1 (P62158) | \nR.EADIDGDGQVNYEEFVQMMTAK | \n
SBP-FLAG-SASH1 affinity purification was performed to identify the peptide sequences of the binding complex of SASH1 protein.
A375 stable cells with ectopic SBP-FLAG-SASH1 expressing were lysed, immunoprecipitated with SBP beads and digested with trypsin. The liquid supernatant was collected, dried, and dissolved in 10% (v/v) acetonitrile and 0.8% formic acid solution. Nanoflow LC-MS/MS analyses were performed to identify the peptides.
Gαs binds to SASH1 and is a central downstream player of p53/POMC cascade. (A) Immunoprecipitation-Western blot (IP-WB) was performed to identify the interactions between GFP-SASH1 and endogenous Gαs in HEK-293T cells. pEGFP-C3-SASH1-recombined vectors were introduced into HEK-293T cells. At 24 h after transfection, GFP-SASH1 was immunoprecipitated and the associated endogenous Gαs was identified by immunoblot analysis using a Gαs antibody. (B) Exogenous Gαs binds to exogenous SASH1. pEGFP-C3-Gαs and pBABE-puro-Flag-SASH1 vectors were co-introduced into HEK-293T cells. At 36 h after transfection, exogenous SASH1 (Flag-SASH1) was immunoprecipitated and the associated GFP-Gas was detected by western blot analysis using an anti-GFP antibody. At 36-h post-transfection, Flag-SASH1 was immunoprecipitated and the associated exogenous Gαs (GFP-Gas) was detected by immuoblot using an anti-GFP antibody. (C) and (D) P53, POMC, and SASH1 are essential to the activation of Gαs. HA-p53, myc-POMC, and GFP-SASH1, respectively, according to different manners of combination were introduced into HEK-293 cells and NHEMs. After 36 h after transfection, immunoblotting was performed to detect the protein levels in two normal cells along with GAPDH as loading control. (E) Exogenous Gαs (GFP-Gαs) is induced by exogenous p53 (HA-p53). HEK-293 cells were transfected with HA-p53 and GFP-Gαs. At 36-h post-transfection, the transfected HEK-293 cells were lysed and subjected to western blot analyses. GFP-Gαs was induced by gradually increased amounts of HA-p53. (F) Exogenous Gαs (GFP-Gαs) is induced by exogenous SASH1 (GFP-Gαs). GFP-Gαs and GFP-SASH1 were transfected into HEK-293T cells. GFP-Gαs was induced by gradually increased doses of GFP-SASH1. (G) and (H) Exogenous POMC (myc-POMC) was induced by increased dose of exogenous p53 in HEK-293T cells and NHEMs. Different amounts of HA-p53 vector and a certain amounts of myc-POMC vector were introduced into HEK-293T cell for expression. Exogenous POMC RNA levels were quantified by quantitative RT-PCR and normalized to GAPDH. The expression of HA-p53 and myc-POMC was analyzed by immunoblot using GAPDH as loading control.
Gαs mediates cAMP production in melanocytes which is stimulated by α-MSH and melanocortins [21] and our study here shows that Gαs is associated with SASH1. Hence, we examine whether Gαs is required for the induction of SASH1 and how Gαs mediates SASH1 expression, we introduced exogenous p53, POMC, Gαs, and SASH1 gene into HEK-293T and NHEMs (normal human epithelial melanocytes) to assess the effects of p53 and POMC on Gαs. Exogenous Gαs was induced in the co-existence of exogenous p53 and POMC (Figure 4C lane 5 and Figure 4D lane 5) and both inducements of exogenous Gαs and exogenous SASH1 were observed in the co-existence of exogenous p53 and POMC in two types of normal cells (Figure 4C lane 6 and Figure 4D lane 6). Meanwhile, in the presence of GFP-SASH1, GFP-Gαs was also induced (Figure 4C lane 4 and Figure 4Dlane 4), which indicated that SASH1 is necessary for activation of GFP-Gαs. And immunoblot showed that Gαs was identified to be induced by exogenous p53 and SASH1 (Figure 4E and F). Our results also demonstrated that POMC was mediated by p53 in HEK-293T and melanocytes, which were consistent with previous conclusions [1] (Figure 4G and H).
\nTo confirm the fact that POMC, p53, and Gαs are needed for the induction of SASH1 and exogenous POMC, p53, Gαs, and SASH1 were transfected into HEK-293T cells and followed by silence of Gαs and POMC by two specific pairs of siRNA, respectively. As identified in HEK-293 cells, deletion of Gαs gene directly induced significant reduction of SASH1 (Figure 5C and D). Deletion of POMC by siRNA resulted in the downregulation of Gαs and SASH1 (Figure 5E and F). Taken above, it is believed that Gαs serves as a pivotal downstream of p53/POMC cascade and SASH1 is regulated by a novel p53/POMC/Gαs cascade.
\nExpression of SASH1 was mediated by a novel p53/POMC/Gαs/SASH1 signal cascade. (A) and (B) Endogenous and exogenous SASH1 (GFP-Gαs) are both induced by Gαs. HEK-293T cells were transfected with gradually increasing doses of exogenous Gαs and exogenous SASH1, or only different amounts of exogenous Gαs. The protein levels of endogenous or exogenous SASH1 were assessed by immunoblot. (C) and (D) Gαs is essential for the induction of SASH1. Exogenous Gαs, POMC, and SASH1 as well as increasing doses of HA-p53 according to different combinations were transfected into HEK-293 cells. Among the transfected HEK-293 cells, two groups of cells were afterward transfected with two pairs of effective Gαs siRNAs and negative control (NC) siRNA. The corresponding protein levels were assessed by western blot. (E) and (F) POMC is essential for the induction of SASH1 and Gαs. HEK-293 cells were transfected with GFP-Gαs, myc-POMC, and GFP-SASH1 as well as increasing dose of HA-p53 according to different manner of combinations. Among the transfected HEK-293 cells, two groups of HEK-293 cells were later silenced with two pairs of effective POMC siRNAs and NC siRNA.
To reveal the phenomenon that P53 physiologically triggers SASH1, discarded normal human foreskin samples were irradiated to gradually increased dose of UV and stained for the histological analyses of p53, ACTH/POMC, and SASH1. Immunohistochemical (IHC) analyses demonstrated p53 is quickly induced in basal layers at the 0.5 J/cm2 dose of UV exposure. The quick inducement of SASH1 and POMC/ACTH at UV irradiation 1.0 J/cm2 dose in melanocytes is followed closely by p53 up-regulation (Figure 6A). Previous study had indicated that up-regulated POMC gene is induced at both protein and mRNA levels following UV exposure of skin [22, 23]. Followed the previous reports [1], a 100 J/m2 UVB dose was administered in this study. The 100 J/m2 UVB dose equates to the standard erythema dose (SED), which is commonly used as a measure of sunlight [24]. Therefore, both endogenous p53 and SASH1 protein levels in HEK-293T cells and NHEMs with ectopic exogenous POMC after UV irradiation were assessed by immunoblot. Expression of exogenous POMC and endogenous SASH1 was markedly induced by 6 h after UV irradiation, which accords with its known stabilization by UV in NHEMs. At 24 h, in NHEMs, UV irradiation maximally promoted the expression of POMC, p53, and SASH1 protein (Figure 6B). Similar induction of exogenous POMC and endogenous p53 and SASH1 was detected in HEK-293T cells after UV irradiation (Figure 6C). Hence, it is believed that both POMC and SASH1 serve as novel downstream players which respond to p53 inducement by UV irradiation.
\nSASH1 is induced physiologically by p53 after UV irradiation. (A) Upon UV irradiation or without UV irradiation, immunohistochemistry analyses of p53, POMC, and SASH1 in human foreskin indicated that p53 is activated by UV-induced-increase of POMC and SASH1. The human foreskin tissues obtained from a 14-year-old boy were irradiated at different doses of UV intensity, then fixed in 10% formalin and embedded in paraffin for immunohistochemical analyses. Scale bar: 20 μm. The representative positive cells of p53, ACTH, and SASH1 were donated by red arrows. (B) and (C) NHEMs and HEK-293T cells with ectopic exogenous POMC (myc-POMC) expression were irradiated with UV irradiation (100 mJ/cm2) and recovered for the indicated times. Transfected cells were lysed and at different time-points after irradiation as indicated. Western blot was used to detect the protein levels of endogenous p53, endogenous SASH1, and exogenous POMC along with GAPDH or beta-tubulin as a loading control.
To assess whether p53 is required for the inducement of SASH1, exogenous p53, and POMC gene were transfected into HEK-293T and NHEMs to test the induction of p53 and POMC to SASH1. In NHEMs and HEK-293T cells with ectopic of POMC (myc-POMC) in NHEMs and HEK-293T cells, exogenous SASH1 were induced to up-regulate by p53 (Figure 7). Increased protein levels of exogenous SASH1 was induced by increasing amounts of exogenous p53 in two normal cells (Figure 8A and B). On the contrary, exogenous p53 (HA-p53) was also triggered by increasing amounts of exogenous SASH1 (Figure 8C and D). The induction of exogenous SASH1 to endogenous p53 was also identified. It has been documented that, in two types of normal cells, increased endogenous p53 was induced by increasing doses of exogenous SASH1 (Figure 8E and F).
\nExogenous p53 triggers expression of SASH1. (A) Exogenous p53 caused up-regulation of exogenous SASH1 in HEK-293T cells. HA-TP53, GFP-SASH1, and myc-POMC were transfected into HEK-293T cells for transient expression. Cells were lysed in 0.5% NP40 buffer containing protease inhibitors and subjected to western blot along with GAPDH as loading control. (B) Exogenous p53 caused up-regulation of exogenous SASH1 in NHEMs.
There is a reciprocal induction between p53 and SASH1 in normal cells. (A) and (B) Exogenous SASH1 was triggered by exogenous p53 (HA-p53) in a dose-dependent manner. Different amounts of HA-TP53 plasmid were introduced into HEK-293T cells and NHEMs as indicated. After 48-h post-transfection or transfection, total RNA of HEK-293T cells and NHEMs was extracted and exogenous SASH1 RNA levels were assessed by quantitative RT-PCR and normalized to GAPDH. Expression of exogenous p53 protein and SASH1 were analyzed by western blot along with GAPDH as a loading control. (C) and (D) Protein and RNA levels of exogenous p53 were promoted by exogenous SASH1 promotes expression. Different amounts of GFP-SASH1 plasmid and a certain amount of exogenous p53 were transfected to HEK-293T cells and NHEMs cells. As revealed by QRT-PCR and western blot, enhanced expression of exogenous TP53 was induced by increasing amounts of GFP-SASH1. (E) and (F) Increased endogenous p53 was induced by exogenous SASH1. Different amounts of GFP-SASH1 were transfected in to HEK-293T cells and NHEMs. At 36 h after transfection, cells were lysed and subjected to western blot to analyze the expression of GFP-SASH1 with GAPDH as loading control. Results are the representative of three independent experiments. (G) A novel reciprocal induction of p53 and SASH1 is mediated by an autoregulatory p53/POMC/Gαs/SASH1 loop. p53 is activated by different types of stress, which fosters POMC, Gαs, and SASH1 successively. The inducement of SASH1 by p53/POMC/Gαs cascade promotes the up-regulation p53 in nucleus, then induced nucleic p53 conversely activates POMC/Gαs/SASH1 cascade, which consists an autoregulatory p53/POMC/Gαs/SASH1 loop.
Since SASH1 is mediated by p53, we want to investigate whether there is a direct relationship between SASH1 and p53. As indicated in Figure 9A and B, HA-p53 did not bind to GFP-SASH. So, we tested the proximal 1 kb promoter region of the SASH1 gene to find the consensus transcription-factor-binding elements that are conserved between human, rat, and mouse. Among the various consensus elements searched for, p53 gene was remarkable. A most possible p53-binding site, sequence of which is “tgcccaagctttcacacttgttt” was identified in the SASH1 5′ flanking region about 550 bp upstream of the transcription initiation site in humans (Figure 9C). So, three synthesized probes were used to investigate the associations of p53 protein with SASH1 gene promoter. However, analyses of electrophoretic mobility shift assay (EMSA) revealed that there was no p53 protein bind the promoter region of the SASH1 gene (Figure 9D).
\np53 is not associated with SASH1 and SASH1 is not transcriptionally regulated by p53. (A) and (B) HEK-293T cells were co-transfected with the pEGFP-C3-SASH1 and Pcdna 3.0-HA-p53 vectors. At 24-h post-transfection, GFP-SASH1 was immunoprecipitated and the associated HA-p53 was detected by western blot analysis using an anti-HA antibody. Similarly, HA-p53 was immunoprecipitated and the associated GFP-SASH was detected by western blot analysis using an anti-GFP antibody. (C) Showed a schematic representation of the SASH1 locus, which indicates location of a p53-binding consensus sequence. (D) EMSA analyses demonstrated that there was none of among three probes of SASH1 gene promoter to bind p53 recombined protein.
In summary, it is believed that SASH is regulated by the p53/POMC/α-MSH/Gαs signal cascade and p53/POMC/α-MSH/Gαs cascade and SASH1 constitute a novel autoregulatory loop. The p53/POMC/α-MSH/Gαs/SASH1 regulatory loop acts as an auto-feedback circuit to regulate the p53-SASH1 reciprocal inducement (Figure 8G).
\nSASH1 up-regulation is mediated by SASH1 mutations, which is unfathomable enigma to us for lone time. Therefore, HEK-293T cells and NHEMs were transfected with wild-type or mutant SASH1 (wt SASH1 or mut SASH1), exogenous p53 and exogenous POMC to assess the effects of SASH1 mutations on p53 and POMC. As demonstrated in Figure 10A and B, increased expression of p53 and POMC was induced by SASH1 mutations. The effects of SASH1 mutations on endogenous p53 at protein level were also assessed. Increased endogenous p53 was also induced by mutated SASH1 (Figure 10C and D). In order to identify that p53 is induced by SASH1 mutations in vivo, immunostaining of p53 in the affected epithelial tissues with SASH1 Y551D mutation was performed. IHC analyses indicated that more nucleic expression of p53 in epithelial tissues and more p53-positive cells in affected epithelia layers were induced by SASH1 Y551D mutation (Figure 10E).
\np53 and POMC are induced to be increased SASH1 mutations. (A) and (B) Up-regulated SASH1 induced by SASH1 mutations promotes the expression of exogenous p53 and exogenous POMC in HEK-293T cells and NHEMs. Wt and mutant SASH1, exogenous p53 and exogenous POMC were introduced into HEK-293T cells and NHEMs. At 48-h post-transfection, immunoblot were performed to detect the corresponding protein levels. (C) As identified by IHC analyses, high expression of endogenous p53 was induced by Y551D-SASH1 mutation and more p53-positive epithelial cells were detected in the affected epithelial tissues. Affected epithelial tissues with Y551D SASH1 mutation from pedigree I as well as normal epithelial tissues were fixed and embedded in paraffin for immunohistochemistry detection. Scale bar: 20 μm. The representative positive cells of p53 are donated by red arrows. (D) and (E) Western blot indicated that increased endogenous p53 was induced by SASH1 mutations in HEK-293 cells and NHEMs.
All of these indicate that not only SASH1 is positively regulated by the p53/POMC/α-MSH/Gαs/SASH1 autoregulatory loop, but also SASH1 mutations serve more as molecular rheostats rather than an on-off switch to control this regulatory loop.
\nSince there is a SASH1-p53 autoregulatory loop, the changes of downstream partners of SASH1 need to be tested. Therefore, we further identified the effects of mutated SASH1 on the protein levels of matrix proteins and transport proteins. Enhanced expression of TYRP1, Rab 27a, Pmel17, and tyrosinase in SK-MEL-28 cells, a pigmented melanoma cell line and NHEMs was significantly induced by SASH1 mutations (Figure 11A and B). QRT-PCR also indicated that Pmel17, TYRP1, and Rab 27a were up-regulated by mutations of SASH1 in SK-MEL-28 stable cells (Figure 11C). Pmel17, TYRP1, and Rab 27a was heterogeneously distributed in the epithelial cells in the tissues of DUH-affected individuals as demonstrated by IHC analyses (Figure 11D and E). Increased levels of melanogenesis molecules were observed in some hyperpigmentation areas in the affected epithelial layers. In the hyperpigmentation plaques, the superfluous production and secretion of melanin was clearly presented in the basal layers and in the suprabasal layers of the affected epidermal as (Figure 11D).
\nIncreased production of melanogenic components and heterogeneous distribution of melanin in vivo were induced by SASH1 mutations. (A) Up-regulation of melanogenic components including TYRP1, Pmel17, tyrosinase, and Rab 27a were induced by SASH1 mutations in stable SK-MEL-28 cells. (B) Up-regulation of Rab 27a and tyrosinase was also induced by SASH1 mutations. The SASH1 gene (wt and mutant) was introduced into NHEMs and western blot was performed to determine the effect of SASH1 mutations on melanogenic components. (C) As identified by QRT-PCR, up-regulation of Pmel17, TYRP1, and Rab 27a in stable SK-MEL-28 cells was induced by SASH1 mutations (n = 4, mean ± standard error). (D) As identified by immunochemistry detection, heterogeneous distribution of Rab 27a and melanin were observed in the epithelial layers of the affected individuals. (E) Immunochemical analyses indicated that expression of Pmel17 and TYRP1 was heterogeneously distributed in all of the epithelial layers of the epidermal tissues from the DUH-affected individuals. Pmel17, TYRP1, and Rab 27a: 400× magnification, bar = 20 μm; melanin: 1000 magnification. Scale bar: 20 μm. The representative positive cells of Rab 27a, Pmel17, TYRP1, and melanin were indicated by red arrows.
Our study reveals that a novel p53-SASH1 reciprocal induction triggers pigmentation of skin through an autoregulatory p53/POMC/α-MSH/Gαs/SASH1 loop. SASH1 mutations enhance SASH1-mediated induction of p53 and POMC. POMC is induced by p53 overexpression and resulted in UV-dependent hyperpigmentation UV-independent pathological hyperpigmentation [1]. Our work indicates that POMC up-regulation is induced by SASH1 mutations, which ultimately results in the pathological hyperpigmentations of affected DUH individuals. These data indicate that SASH1 activation induced by mutations in melanocytes acts as a “UV sensor/effector” for skin pigmentation or SASH1 mutations-mediated up-regulation is the “chief criminal” of pathological hyperpigmentation of DUH, and its underlying mechanistic role is SASH1-p53 reciprocal inducement. Our data indicate that the definitions of the positive feedback p53/POMC/α-MSH/Gαs/SASH1 loop help us to recognize an important linkage between the p53 pathway and MC1R pathway by SASH1.
\nRecently, a c.1067T>C (p.Leu356Pro) mutation in exon 3 of ABCB6 (ATP-binding cassette subfamily B, member 6) was found in a large five-generation Chinese family with DUH family. Two additional missense mutations, c.508A>G (p.Ser170Gly) in exon 1 and c.1736G>A (p.Gly579Glu) in exon 12 of ABCB6 were found in two out of six patients using sporadic DUH patients [25]. Ac.1663C>A, (p.Gln555Lys) missense mutation in ABCB6 was identified in a Chinese family with typical features of autosomal dominant DUH. Two deletion mutations (g.776 delC, c.459 delC) in ABCB6 were found in an unrelated sporadic affected individual [26]. In addition, missense mutations in ABCB6 were also found in the sporadic affected DUH individuals [27, 28]. Silence of ABCB6 by siRNA destroyed PMEL amyloidogenesis in early melanosomes and resulted in aberrant increase of multilamellar aggregates in pigmented melanosomes. In the retinal pigment epithelium of ABCB6 knockout mice, morphological analysis indicated an obvious decrease of melanosome numbers [29]. All of these sequencing results and functional analyses of causing genes responsible for DUH indicate there exist novel pathogenicity genes and novel gene variations which is responsible for pathogenesis of DUH or there exists novel subtype of DUH.
\nThe transcriptional network of p53-responsive genes produces proteins that interact with a large number of other signal transduction pathways in the cell and a number of positive and negative autoregulatory feedback loops act upon the p53 response [30]. Feedback loops of p53 and p53-responsive genes provide a means to connect the p53 pathway with other signal transduction pathways and coordinate the cellular signals for growth and division [30]. Our findings suggest that SASH1 serves as a “Hinge” to connect p53/POMC/α-MSH pathway with MC1R/Gαs/cAMP/PKA cascade to form an autoregulatory p53/POMC/Gαs/SASH1 circuit to mediate the melanogenesis process [18, 31].
\nMost recently, SASH1 is showed to involve in autosomal-dominant lentiginous [32] and autosomal-recessive SASH1 variants (c.1849G>A; p.Glu617Lys), which are associated with a new genodermatosis with a pigmentation defects, palmoplantar keratoderma and skin carcinoma and SASH1 is first reported to be predisposed to skin cancer [33]. Dyschromatosis universalis hereditaria (DUH) is a clinically heterogeneous disorder that presents as generalized mottled pigmentation and was first reported by Ichigawa and Hiraga in 1933 [7]. Stuhrmann and colleagues identified the first locus responsible for autosomal-recessive DUH, and this findings is consistent with recent evidence demonstrating that DSH and DUH are genetically distinct disorders [34]. Zhang et al. mapped the causative gene of DSH to 1q11-1q21 and found that a novel mutation of a heterozygous nucleotide A → G at position 2879 in exon 10 of the DSRAD gene is involved in DSH [35]. Subsequent research on dyspigmentation has demonstrated that the pathogenic genetic variant that causes DSH is localized to the DSRAD gene on chromosome 1q [15, 36, 37, 38, 39, 40, 41]. Expanding Stuhrmann and Nuber’s findings and our own previous work providing photographic evidence of dyschromatosis presenting as large hyperpigmented bodies on DUH-affected individuals [6, 8, 34], we believe that we have discovered the first locus associated with autosomal dominant DUH, identifying SASH1 as the causative gene of autosomal dominant DUH.
\nOur findings first identify the pathological gene of DUH and reveal the pathological mechanism of hyperpigmentation patches of DUH-affected individuals. In addition, our work will enrich the crosstalk of p53 pathway with other transduction pathways in cells and give a new definition of the p53-responsive genes and their associations, which will perfect the p53 programmed responses to stress and pathologic conditions.
\nWe thank Central Laboratory at Yongchuan Hospital, Chongqing Medical University and Clinical Research Center, the Affiliated Hospital, Guizhou Medical University for housing the experiments. This work was supported by the Shanghai Municipal Commission of Science and Technology Program (09DJ1400601), the 973 Program (2010CB529600, 2007CB947300), the Yongchuan Hospital Project, Chongqing Medical University (YJYJ201347), Chongqing Education Commission Project (KJ1400201), and Guizhou’s Introduction Project of Million Talents.
\nNo conflict between the authors.
The chapter text was mainly referred to our article entitled as “A Novel P53/POMC/Gαs/SASH1 Auto-regulatory Feedback Loop Activates Mutated SASH1 to Cause Pathologic Hyper-pigmentation” (Journal of Cellular and Molecular Medicine 2017, 21(4):802-815) which we published in journal of cellular and molecular medicine in April, 2017. In this chapter, we rewrite the chapter text according to the suggestions of reviewers.
\nThe chapter figures were taken from the figures and supplementary figures of our article entitled as “A Novel P53/POMC/Gαs/SASH1 Auto-regulatory Feedback Loop Activates Mutated SASH1 to Cause Pathologic Hyper-pigmentation”. The chapter tables were taken from the supplementary tables of our published article.
\nOur article entitled as “A Novel P53/POMC/Gαs/SASH1 Auto-regulatory Feedback Loop Activates Mutated SASH1 to Cause Pathologic Hyper-pigmentation” is an Open Access article published under the terms of the Creative Commons Attribution License (CC BY). We are allowed to reuse the material without having to obtain permission provided that the original source of publication.
\nABCB6 | ATP-binding cassette subfamily B, member 6 |
ACTH | adrenocorticotropic |
α-MSH | α-melanocyte stimulating hormone |
cAMP | cyclic adenosine monophosphate |
CDS | coding sequence |
CHX | cycloheximide |
DSRAD | double-stranded RNA-specific adenosine deaminase |
DSH | dyschromatosis symmetrica hereditaria |
DUH | dyschromatosis universalis hereditaria |
EMSA | electrophoretic mobility shift assay |
GAPDH | glyceraldehyde phosphate dehydrogenase |
Gαs | guanine nucleotide-binding protein subunit-alpha isoforms short |
GPCR | G-protein-coupled receptor |
GS | Griscelli syndrome |
IF | immunofluorescence |
IHC | immunohistochemical |
LC-MS/MS | liquid chromatograph-mass spectrometer |
LOD | log odds |
LSD | least-significant-difference |
MC1R | melanocortin-1-receptor |
Mitf | microphthalmia-associated transcription factor |
NHEMs | normal human epithelial melanocytes |
PEI | polyethyleneimine |
POMC | pro-opiomelanocortin |
QRT-PCR | quantitative reverse transcriptase polymerase chain reaction |
RT-PCR | reverse transcriptase polymerase chain reaction |
SASH1 | SAM and SH3 domain containing 1 |
SED | standard erythema dose |
TYRP1 | tyrosinase protein1 |
UV | ultraviolet |
Open Access publishing helps remove barriers and allows everyone to access valuable information, but article and book processing charges also exclude talented authors and editors who can’t afford to pay. The goal of our Women in Science program is to charge zero APCs, so none of our authors or editors have to pay for publication.
",metaTitle:"What Does It Cost?",metaDescription:"Open Access publishing helps remove barriers and allows everyone to access valuable information, but article and book processing charges also exclude talented authors and editors who can’t afford to pay. The goal of our Women in Science program is to charge zero APCs, so none of our authors or editors have to pay for publication.",metaKeywords:null,canonicalURL:null,contentRaw:'[{"type":"htmlEditorComponent","content":"We are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor this ambitious program, we’d love to hear from you. Contact Dr. Anke Beck at anke@intechopen.com.
\\n\\nAll of our IntechOpen sponsors are in good company! The research in past IntechOpen books and chapters have been funded by:
\\n\\nWe are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor this ambitious program, we’d love to hear from you. Contact Dr. Anke Beck at anke@intechopen.com.
\n\nAll of our IntechOpen sponsors are in good company! The research in past IntechOpen books and chapters have been funded by:
\n\n