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• W2782804955 abstract "Article8 January 2018free access Source DataTransparent process STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis Rajender K Motiani Corresponding Author Rajender K Motiani [email protected] orcid.org/0000-0002-8971-9008 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Jyoti Tanwar Jyoti Tanwar Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Desingu Ayyappa Raja Desingu Ayyappa Raja Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Ayushi Vashisht Ayushi Vashisht orcid.org/0000-0003-3458-5329 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Shivangi Khanna Shivangi Khanna Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Sachin Sharma Sachin Sharma Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Sonali Srivastava Sonali Srivastava Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Sridhar Sivasubbu Sridhar Sivasubbu Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Vivek T Natarajan Vivek T Natarajan Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Rajesh S Gokhale Corresponding Author Rajesh S Gokhale [email protected] orcid.org/0000-0001-6597-2685 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Rajender K Motiani Corresponding Author Rajender K Motiani [email protected] orcid.org/0000-0002-8971-9008 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Jyoti Tanwar Jyoti Tanwar Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Desingu Ayyappa Raja Desingu Ayyappa Raja Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Ayushi Vashisht Ayushi Vashisht orcid.org/0000-0003-3458-5329 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Shivangi Khanna Shivangi Khanna Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Sachin Sharma Sachin Sharma Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Sonali Srivastava Sonali Srivastava Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Sridhar Sivasubbu Sridhar Sivasubbu Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Vivek T Natarajan Vivek T Natarajan Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Academy of Scientific and Innovative Research, New Delhi, India Search for more papers by this author Rajesh S Gokhale Corresponding Author Rajesh S Gokhale [email protected] orcid.org/0000-0001-6597-2685 Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India Search for more papers by this author Author Information Rajender K Motiani *,1, Jyoti Tanwar1, Desingu Ayyappa Raja1,2, Ayushi Vashisht1, Shivangi Khanna1,2, Sachin Sharma1,2, Sonali Srivastava1, Sridhar Sivasubbu1,2, Vivek T Natarajan1,2 and Rajesh S Gokhale *,1,† 1Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India 2Academy of Scientific and Innovative Research, New Delhi, India †Present address: National Institute of Immunology, New Delhi, India *Corresponding author. Tel: +91 011 29879221; E-mail: [email protected] *Corresponding author. Tel: +91 11 26703545; E-mail: [email protected] The EMBO Journal (2018)37:e97597https://doi.org/10.15252/embj.201797597 See also: J Soboloff et al (March 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Endoplasmic reticulum (ER)–plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca2+ levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca2+ stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo. STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions. Synopsis Interaction of the endoplasmic reticulum (ER) calcium sensor STIM1 with Orai channels at the plasma membrane mediates cellular calcium influx after its depletion from ER stores. New data show that adenylyl cyclase 6 (ADCY6) is an Orai-independent STIM1 interactor at ER–plasma membrane junctions with a critical role in melanogenesis. STIM1 regulates pigmentation independently of Orai channels. α-MSH induces ER Ca2+ release and STIM1 activation that in turn regulates pigmentation. STIM1 interacts with ADCY6 and stimulates its activity to increase cAMP generation and melanogenesis. STIM1 Ser/Pro-rich domain mediates the interaction with ADCY6. Introduction The endoplasmic reticulum (ER) is the largest membrane system in animal cells that forms a continuous network extending from the cellular plasma membrane (PM) to the nuclear envelope (David, 2013; English & Voeltz, 2013). ER is a key biosynthetic hub that produces variety of secretory proteins and lipid species (David, 2013; Stefan et al, 2013) and additionally maintains a large intracellular Ca2+ store. Store-operated Ca2+ entry (SOCE) is a crucial pathway activated at ER-PM junctions (Smyth et al, 2010). SOCE is initiated upon Ca2+ release from the intracellular stores of ER. STIM1, a Ca2+ binding ER membrane protein, senses the decrease in ER Ca2+ levels. Upon loss of Ca2+, STIM1 oligomerizes and interacts with Orai channels at the plasma membrane. This results in opening of the Orai channels leading to Ca2+ influx into the cells (Smyth et al, 2010). Several other interacting partners for STIM1 such as voltage-gated Ca2+ channels (CaV), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and TRP channels have been reported and are known to mediate specific signaling events (Park et al, 2010; Wang et al, 2010; Gruszczynska-Biegala et al, 2016; Shin et al, 2016). Ca2+ and cAMP are the two fundamental secondary messengers that dictate variety of cellular processes. Interestingly, the two regulate one another at multiple levels downstream of various G-coupled receptor signaling modules. Changes in bulk cytosolic Ca2+ are known to regulate cAMP levels through the different isoforms of adenylyl cyclases (ADCYs), which catalyze the conversion of ATP to cAMP (Cooper, 2015). cAMP primarily mediates its effects either by activating protein kinase A (PKA) or through phospholipase C (PLC) involving exchange protein directly activated by cAMP (EPAC) (Schmidt et al, 2001; Cooper, 2015). In a variation to this theme, a recent study proposed that the ER Ca2+ stores could directly regulate cAMP signaling (Lefkimmiatis et al, 2009). While this connects the two major signaling hubs, the molecular and physiological context leading to this crosstalk between ER Ca2+ and cAMP remains to be determined. There are 10 different isoforms of ADCYs, and their expression varies among different cell types. Out of these, nine (ADCY1-9) are plasma membrane-bound whereas ADCY10 is soluble and is present in the cytosol (Halls & Cooper, 2017). Interestingly, ADCY1, 3, and 8 are activated by Ca2+, while ADCY5 and 6 are inhibited by Ca2+ (Cooper, 2015; Halls & Cooper, 2017). Recently, Orai1 was reported to regulate cAMP generation by physically interacting with ADCY8 resulting in its activation and thereby elevation of cAMP (Willoughby et al, 2012). However, the question whether STIM1 can physically interact and activate ADCYs, and the cellular context and consequence of this interplay have remained unanswered. cAMP signaling is the central mediator of melanogenesis programming in melanocytes (D'Orazio & Fisher, 2011). Melanin produced within melanosomes is transferred to neighboring keratinocytes, which provides protection to the skin from UV-induced cellular damage (Natarajan et al, 2014a). One of the major physiological determinants of pigmentation in humans is α-melanocyte-stimulating hormone (αMSH; Videira et al, 2013; Abdel-Malek et al, 2014). αMSH binds to the G-coupled receptor melanocortin-1 receptor (MC1R) and activates melanogenesis via cAMP-PKA-microphthalmia transcription factor (MITF) signaling axis (Levy et al, 2006). MITF is the key transcription factor that regulates both pigmentation and melanocyte proliferation. A variety of signaling pathways converge on this master regulator of pigment cell biology for calibrating melanogenesis and melanocyte proliferation (Levy et al, 2006; Lin & Fisher, 2007; Hartman & Czyz, 2015). While the role of cAMP is well established in melanogenesis process, the significance of Ca2+ signaling in controlling melanogenesis remains poorly understood (Videira et al, 2013). Incidentally, depletion of internal Ca2+ stores is a common physiological outcome of hormone-controlled intracellular signaling (Bootman, 2012). We therefore decided to carefully reexamine the signaling events downstream of αMSH, particularly from the perspective of ER Ca2+ stores and the cAMP signaling. Surprisingly, the role of SOCE components in the signaling events that lead to pigmentation remains poorly understood. An isolated study has implicated a role for Orai1 in endothelin-1-mediated melanogenesis (Stanisz et al, 2012). While STIM1 is expressed in melanocytes (Stanisz et al, 2016), its role in melanogenesis remains hitherto uncharacterized. Here we demonstrate that Ca2+ concentration in ER can directly control cAMP production during αMSH-induced pigmentation, independently of the changes in cytosolic Ca2+. αMSH induces ER Ca2+ release and activates STIM1 oligomerization at ER-PM junctions. While the STIM1 and Orai1 activate SOCE, this influx of Ca2+ primarily governs proliferation and is not involved in pigmentation. STIM1 recruitment at PM activates ADCY6, increasing the cAMP levels and thus forming a novel positive feedback loop that sustains elevated cAMP levels required to signal the induction of pigmentation genes. We identify a critical role of STIM1 S/P-rich domain in ADCY6 interaction and melanogenesis. Further, by studying STIM1 in zebrafish, we substantiate the significance of this protein in pigmentation biology. This elucidation of the physiological circumstances leading to the initiation of the cAMP production through ADCY6-STIM1 interaction could provide new avenues for developing therapeutic strategies for treatment of malignant and pigmentary disorders. Results Transcriptome analysis identifies importance of Ca2+ signaling in melanogenesis Melanogenesis is a complex process wherein melanin pigment is produced in melanosomes by melanocytes. While the classical pathway of αMSH-cAMP-PKA-MITF is known to govern pigmentation and proliferation, a recent study suggested αMSH's role in activating PLCβ/PI(4,5)P2 pathway (Maresca et al, 2012) that is known to induce release of Ca2+ from ER stores. This suggested a distinct possibility of a crosstalk between cAMP and Ca2+ during melanocyte pigmentation. In order to get a broader understanding of the pathways involved in melanogenesis, we performed whole-genome transcriptome profiling of human primary melanocyte. We compared between the melanocyte pigmentation inducer tyrosine and depigmenting agent phenylthiourea (PTU) (Fig 1A). RNA expression analysis revealed differential regulation for the expected melanogenic genes, and surprisingly, both the Ca2+ and cAMP signaling networks emerged among the key enriched pathways (Fig 1B). Although tyrosine and PTU act downstream in the melanogenesis pathway, perturbation of Ca2+ and cAMP pathways, probably through feedback signaling, suggested relevance of these modules in melanogenesis. Figure 1. SOCE is enhanced with increase in pigmentation Pictorial representation of the microarray performed on human primary melanocytes for identification of novel regulators of the pigmentation. Pathway enrichment plot for the differentially regulated signaling pathways upon tyrosine and PTU treatment. Representative Ca2+ imaging trace of Tg-stimulated SOCE measurement in primary human melanocytes. Here, “n” denotes the number of cells in the trace. Representative Ca2+ imaging trace of Tg-stimulated SOCE measurement in B16 cells. Here, “n” denotes the number of cells in the trace. B16 cell pellet pictures of LD day 0, LD day 4, and LD day 7 along with the analysis of melanin concentration in these cells. The error bars indicate SD (N = 3). SOCE was measured on different days of LD cultures, and data for amplitude of SOCE are presented in bar graphs. Data information: Total cells imaged in (F) are reported as “n = x, y” where “x” denotes the number of cells imaged and “y” denotes the number of traces recorded for imaging “x” cells. Data presented in (F) are Mean ± SEM (****P < 0.0001; unpaired Student's t-test was performed for statistical analysis). Download figure Download PowerPoint While cAMP signaling is the central mediator of the melanogenesis programming, the relevance of Ca2+ signaling is not well established. Activation of SOCE at ER-PM junction is one of the key components of cellular Ca2+ homeostasis. In order to understand the role of SOCE in mammalian melanogenesis, we examined SOCE in primary human melanocytes and in mouse B16F10 melanoma (B16) cells. We performed live-cell ratiometric Ca2+ imaging using FURA2 dye that measures cytosolic Ca2+ levels and used classical pharmacological tool thapsigargin (Tg) for rapidly depleting ER Ca2+ levels. A typical Ca2+ imaging trace is shown in Fig 1C for primary melanocytes, where the first peak corresponds to the release of Ca2+ from ER stores. After reaching the steady-state levels, addition of extracellular Ca2+ showed a sharp increase in the relative fluorescence intensity that corresponds to the Ca2+ entry into the cells. Similar trace could be obtained for the depigmented B16 cells (Fig 1D), although the amplitude of SOCE was much smaller for depigmented B16 cells. To examine whether the Ca2+ entry through SOCE is modulated during the melanogenesis, we measured SOCE during the course of pigmentation. We had earlier developed a pigmentation oscillator model with B16 cells, wherein low-density (LD) plating of non-pigmented cells brings about synchronous pigmentation over a period of 7 days (Natarajan et al, 2014b). As measured by the melanin concentration assay, LD day 0 cells are non-pigmented; day 4 cells show intermediate pigment levels while day 7 cells are highly pigmented (Fig 1E). This model is shown to recapitulate the pigmentation responses observed in primary melanocytes (Natarajan et al, 2014b). Overlay of the Ca2+ imaging traces showed dramatic change in the SOCE amplitude with more than twofold increase at day 4 and greater than threefold increase on day 7, in comparison with day 0 cells (Fig 1F). The correlation of SOCE amplitude with increased melanin content suggested a role of Ca2+ signaling in pigmentation biology. We therefore investigated whether the proteins involved in establishing SOCE are also regulated during melanogenesis. STIM1 and Orai1 mediate melanocyte SOCE STIM and Orai proteins are the primary mediators of SOCE. To identify the specific STIM and Orai homologs that mediate SOCE in B16 cells, we standardized siRNA silencing of STIM and Orai genes (STIM1, STIM2, Orai1, Orai2, and Orai3). The transfection of B16 cells with siRNAs showed significant decrease (60–70%) in the levels of respective target mRNA 72 h post-transfection (Appendix Fig S1A). The measurement of Ca2+ fluxes and their comparative analysis showed no significant differences for ER Ca2+ release in any of the five knockdowns (Fig 2A and Appendix Fig S1B). However, a marked decrease was seen in the SOCE amplitude with siSTIM1 and siOrai1 (Fig 2A and B). To substantiate these findings, we generated lentiviral-transduced shSTIM1 and shOrai1 B16 stable cell lines (Appendix Fig S2A). In both these lines, we obtained around 75% decrease in the respective mRNA levels (Appendix Fig S2B and C) and 55–60% reduction in their protein levels (Appendix Fig S2D and E). The Ca2+ imaging studies in the two stable lines showed no significant differences in the ER Ca2+ release (Appendix Fig S1C) and 50% reduction in the amplitude of SOCE in comparison with shControl B16 stable line (Fig 2C and D). This suggested that STIM1 and Orai1 mediate SOCE in melanocytes. Figure 2. STIM1 and Orai1 mediate SOCE in melanocytes A. Representative SOCE traces of all five STIM and Orai homologs' knockdowns and non-targeting siRNA control where “n” denotes the number of cells in that particular trace. B. The amplitude of SOCE was calculated from a number of experiments, and data are presented in bar graphs. C. Representative Ca2+ imaging trace of B16 stable cell lines; control shLuciferase, shSTIM1 and shOrai1 stables where “n” denotes the number of cells in that particular trace. D. The amplitude of SOCE was calculated from a number of experiments, and data are presented in bar graphs. E, F. Western blot analysis showing an increase in STIM1 protein expression on LD day 7 in comparison with day 0 whereas expression of Orai1 remained largely unchanged. Data information: Data presented in (B and D) are Mean ± SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student's t-test was performed for statistical analysis). The number of cells is reported as “n = x, y” where “x” denotes the total number of cells imaged and “y” denotes number of traces recorded for imaging “x” cells. Source data are available online for this figure. Source Data for Figure 2 [embj201797597-sup-0004-SDataFig2.zip] Download figure Download PowerPoint We then examined the expression levels of STIM1 and Orai1 during B16 LD pigmentation. About threefold increase in STIM1 protein expression on LD day 7 could be noted, as compared to the day 0 cells (Fig 2E). Orai1 levels however showed no significant difference (Fig 2F). This was somewhat surprising since the increased Ca2+ influx through SOCE would require formation of cognate STIM1-Orai1 complexes. Since Ca2+ is ubiquitously known to play an important role in cellular proliferation, we therefore deliberated whether the observed SOCE increase in B16 pigmentation model is an outcome of increased proliferative rates. STIM1 and Orai1 regulate melanocyte proliferation and melanoma growth The central transcription factor of melanocyte MITF is known to regulate both cellular proliferation and pigmentation, and thus, two pathways could be subtly regulated through a common node. Cellular proliferation assays with STIM1 and Orai1 knockdown showed 25–30% reduction in proliferation of B16 cells at 48 and 72 h post-transfection (Fig 3A). The knockdown of MITF resulted in 40% decrease in B16 proliferation. Orai2 and Orai3 silencing showed no significant changes, while STIM2 knockdown enhanced B16 proliferation by approximately 10% (Fig 3A). The proliferation effects for STIM1 and Orai1 were also studied in primary human melanocytes. Primary melanocytes are terminally differentiated and possess weak proliferative capabilities, and therefore, external stimuli of αMSH were used in this study. We achieved about 50% knockdown of STIM1 and Orai1 in primary melanocytes (Fig EV2A and B). Silencing of both STIM1 and Orai1 reduced αMSH-induced melanocyte proliferation by 35–40% (Fig 3B). For corroborating the role of STIM1 and Orai1 in proliferation, we examined the growth of melanoma tumors after subcutaneous injections of shLuciferase control and shSTIM1 or shOrai1 B16 stable cell lines in C57Bl/6 mice. Interestingly, previous reports implicate STIM1 and Orai1 in melanoma metastasis (Sun et al, 2014; Hooper et al, 2016), but their role in melanoma growth remained largely uninvestigated. Post-injections, we followed melanoma tumor development for 15 days by measuring tumor volume (on every alternate day). As shown in Fig 3C, knockdown of both STIM1 and Orai1 drastically reduced melanoma growth. Similarly, the mean melanoma weight at the end of study was also significantly reduced with both shSTIM1 and shOrai1 B16 stables (Fig 3D). Collectively, these studies suggest that STIM1 and Orai1 regulate melanocyte proliferation and melanoma development in vivo. Figure 3. STIM1 regulates melanocyte proliferation and pigmentation while Orai1 only mediates proliferation B16 cell proliferation upon knockdown of STIM and Orai homologs 48 and 72 h post-transfection with siRNAs (N = 3). αMSH-stimulated primary human melanocyte proliferation post-siRNAs transfections (N = 3). B16 melanoma tumor volumes upon subcutaneous injection of B16 stable cell lines in C57Bl/6 mice (five mice/group). Tumor weights in the experimental groups after sacrificing mice on the 15th day post-tumor cell injection (N = 5). Melanin content assay performed on the siRNA-transfected cells for quantifying the affect on LD pigmentation upon all five STIM and Orai homologs' silencing (N = 3). Representative bright-field images of shLuciferase, shSTIM1, and shOrai1 B16 stable cells on LD day 7. Data information: Data represented are Mean ± SEM (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student's t-test was performed; one-way ANOVA was performed for tumor volume analysis). Download figure Download PowerPoint STIM1 but not Orai channels mediate pigmentation Having established STIM1 and Orai1 role in proliferation, we next examined their involvement in pigmentation process. The B16 LD pigmentation levels upon knockdown of STIM and Orai homologs were compared to the levels observed in case of non-targeting control siRNA. The targeted siRNA screening suggested that STIM1 silencing decreases melanogenesis, while knockdown of STIM2 and all the three Orai homologs showed no difference in the levels of melanin formation (Fig 3E). STIM1 silencing resulted in ~35% decrease in melanin content on LD day 7 while Orai1 silencing did not affect melanogenesis (Figs 3E and EV1A and B). The control MITF knockdown resulted in 60% decrease in melanin levels (Figs 3E and EV1B). To provide confidence to the siRNA-mediated knockdown specificity, we expressed human-mCherry STIM1 in siSTIM1 cells. Western blot analysis showed expression of human-mCherry STIM1 in these cells (Fig EV1C). The LD day 7 cells expressing human-mCherry STIM1 along with siSTIM1 indeed showed melanin content comparable to control cells (Fig EV1C). Further, B16 LD pigmentation experiments with shSTIM1 and shOrai1 stable cells also showed a clear decrease in pigmentation level in the bright-field images of LD day 7 cells with STIM1 knockdown. The Orai1 knockdown cells however showed comparable pigmentation to the control shRNA stable cells (Fig 3F). The GFP expression, used as control, showed similar lentiviral transduction efficiency for all the three conditions (Fig EV1D). Together, these studies provided an interesting perspective of Orai1-independent role of STIM1 in melanogenesis. To further establish this novel finding, we carefully examined the role of STIM1 in primary melanocytes and also in the zebrafish model system. Click here to expand this figure. Figure EV1. STIM1 but not Orai1 regulates melanogenesis Western blots demonstrating siRNA-based knockdown of STIM1 and Orai1. Representative cell pellets of LD day 7 upon STIM1, Orai1, and MITF silencing along with melanin content analysis (N = 3). Western blot for examining mCherry-STIM1 expression in B16 cells and melanin content analysis for evaluating rescue of pigmentation loss with mCherry-STIM1 (N = 3). Lentiviral-transduced B16 stable cell lines used for LD melanogenesis assay showing comparable transduction efficiency. Melanin content analysis in the unstimulated LD cells and upon αMSH stimulation in LD cells transfected with siNT, siSTIM1, siOrai1, or siMITF (N = 3). Data information: Data represented are Mean ± SEM (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student's t-test). Download figure Download PowerPoint αMSH-induced pigmentation requires STIM1 Using the physiological stimuli αMSH, we monitored the significance of STIM1 and Orai1 in primary melanocytes and B16 pigmentation. We observed significant decrease in the αMSH-induced melanin generation with STIM1 knockdown and not with Orai1 silencing in the primary human melanocytes (Fig EV2C and D). The analysis of key melanogenic enzymes in the primary melanocytes also substantiated these findings, wherein tyrosinase activity as well as the levels of dopachrome tautomerase (DCT) was substantially reduced upon STIM1 but not Orai1 silencing (Fig EV2E). Similar data for STIM1 and Orai1 knockdown could be noted in B16 LD cells with αMSH stimulation. While Orai knockdown shows similar melanin content to control cells, there was more than 40% decrease for STIM1 knockdown cells (Fig EV1E). Click here to expand this figure. Figure EV2. STIM1 but not Orai1 regulates αMSH-induced primary human melanocyte pigmentation qRT–PCR analysis of STIM1 48 h post-transfection with STIM1 siRNA (N = 3). qRT–PCR analysis of Orai1 48 h post-transfection with Orai1 siRNA (N = 3). Primary human melanocyte pellets showing the extent of αMSH-driven pigmentation upon STIM1 or Orai1 silencing in comparison with siNT control. Melanin content analysis evaluating changes in αMSH-induced pigmentation upon STIM1 and Orai1 silencing (N = 3). Western blots and gel images of tyrosinase activity (DOPA assay) in primary human melanocytes transfected with either control siRNA or STIM1/Orai1 siRNA. Data information: Data represented are Mean ± SEM (*P < 0.05; **P < 0.01, ***P < 0.001; unpaired Student's t-test). Download figure Download PowerPoint We then examined the role of STIM1 in regulating pigmentation in vivo by using zebrafish system. The melanogenesis program is broadly conserved across vertebrates and owing to translucency of zebrafish embryos; the pigmentation can be easily monitored visually and quantified with microscopic analysis (Kelsh et al, 1996). Zebrafish contains two STIM1 paralogs namely zSTIM1a and zSTIM1b, and we followed changes in pigmentation by using morpholino-based knockdown strategy (Bill et al, 2009). Substantial reduction in the pigmented black-colored melanophores (melanocyte equivalents in zebrafish) could be observed in zSTIM1a morphants in comparison with the control morphants at 36 hpf (hours post-fertilization) (Fig 4A). This phenotype was observed in more than 70% embryos (Fig 4B). However, the zSTIM1b morpholino injections showed no perturbation in zebrafish pigment (Fig 4A and B). Since morpholinos are injected at single-cell stage, the loss of pigmented melanophores in zSTIM1a morphants could be a consequence of the developmental arrest or reduced pigment synthesis. The precursor pigment cells, melanoblasts, can be monitored by the presence of MITF, while the melanophores can be studied by the expression of tyrosinase-related protein 1 (TyRP1). We therefore generat" @default.
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• W2782804955 title "<scp>STIM</scp> 1 activation of adenylyl cyclase 6 connects Ca ²⁺ and <scp>cAMP</scp> signaling during melanogenesis" @default.
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