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. 2018 Mar 1;37(5):e97597.
doi: 10.15252/embj.201797597. Epub 2018 Jan 8.

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis

Rajender K Motiani  1 , Jyoti Tanwar  2 , Desingu Ayyappa Raja  2   3 , Ayushi Vashisht  2 , Shivangi Khanna  2   3 , Sachin Sharma  2   3 , Sonali Srivastava  2 , Sridhar Sivasubbu  2   3 , Vivek T Natarajan  2   3 , Rajesh S Gokhale  1
Affiliations

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis

Rajender K Motiani et al. EMBO J. .

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.

Keywords: cAMP; ADCY6; Orai1; STIM1; pigmentation.

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Figures

Figure 1
Figure 1. SOCE is enhanced with increase in pigmentation

  1. Pictorial representation of the microarray performed on human primary melanocytes for identification of novel regulators of the pigmentation.

  2. Pathway enrichment plot for the differentially regulated signaling pathways upon tyrosine and PTU treatment.

  3. Representative Ca2+ imaging trace of Tg‐stimulated SOCE measurement in primary human melanocytes. Here, “n” denotes the number of cells in the trace.

  4. Representative Ca2+ imaging trace of Tg‐stimulated SOCE measurement in B16 cells. Here, “n” denotes the number of cells in the trace.

  5. 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).

  6. 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).
Figure 2
Figure 2. STIM1 and Orai1 mediate SOCE in melanocytes
  1. 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.

  2. B

    The amplitude of SOCE was calculated from a number of experiments, and data are presented in bar graphs.

  3. 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.

  4. D

    The amplitude of SOCE was calculated from a number of experiments, and data are presented in bar graphs.

  5. 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.
Figure 3
Figure 3. STIM1 regulates melanocyte proliferation and pigmentation while Orai1 only mediates proliferation

  1. B16 cell proliferation upon knockdown of STIM and Orai homologs 48 and 72 h post‐transfection with siRNAs (N = 3).

  2. αMSH‐stimulated primary human melanocyte proliferation post‐siRNAs transfections (N = 3).

  3. B16 melanoma tumor volumes upon subcutaneous injection of B16 stable cell lines in C57Bl/6 mice (five mice/group).

  4. Tumor weights in the experimental groups after sacrificing mice on the 15th day post‐tumor cell injection (N = 5).

  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).

  6. 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).
Figure EV1
Figure EV1. STIM1 but not Orai1 regulates melanogenesis

  1. Western blots demonstrating siRNA‐based knockdown of STIM1 and Orai1.

  2. Representative cell pellets of LD day 7 upon STIM1, Orai1, and MITF silencing along with melanin content analysis (N = 3).

  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).

  4. Lentiviral‐transduced B16 stable cell lines used for LD melanogenesis assay showing comparable transduction efficiency.

  5. 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).
Figure EV2
Figure EV2. STIM1 but not Orai1 regulates αMSH‐induced primary human melanocyte pigmentation

  1. qRT–PCR analysis of STIM1 48 h post‐transfection with STIM1 siRNA (N = 3).

  2. qRT–PCR analysis of Orai1 48 h post‐transfection with Orai1 siRNA (N = 3).

  3. Primary human melanocyte pellets showing the extent of αMSH‐driven pigmentation upon STIM1 or Orai1 silencing in comparison with siNT control.

  4. Melanin content analysis evaluating changes in αMSH‐induced pigmentation upon STIM1 and Orai1 silencing (N = 3).

  5. 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).
Figure 4
Figure 4. STIM1 plays a critical role in pigmentation in vivo

  1. Representative images of wild‐type zebrafish embryos injected with either control morpholino or morpholino targeting zebrafish STIM1a or zebrafish STIM1b. Arrows indicate zebrafish melanophores.

  2. The pigmentation phenotype analyzed in over 200‐zebrafish embryos/condition from three independent sets of injections and data are plotted in bar graphs.

  3. Images of ftyrp1:GFP transgenic zebrafish line injected with either control morpholino or STIM1a morpholino showing GFP‐positive differentiated melanophores at 48 hpf.

  4. Imaging FACS analysis showing the numbers of differentiated melanophores in control and STIM1a morphants (N = 3).

  5. Representative images of whole embryo in situ hybridization (WISH) for DCT and tyrosinase (tyr). Arrows points to WISH patterns in zebrafish embryos.

  6. Bar graphs with quantification of in situ data presented in panel (E).

Data information: Data presented in (D) are Mean ± SD (one‐way ANOVA was performed for statistical analysis).
Figure 5
Figure 5. αMSH mobilizes ER calcium

  1. In the absence of extracellular Ca2+, 5 μM αMSH treatment leads to rise in cytosolic Ca2+ levels in primary human melanocytes. Here, “n” corresponds to the number of cells imaged.

  2. In the absence of extracellular Ca2+, αMSH induces increase in cytosolic Ca2+ in B16 cells.

  3. αMSH is not able to induce cytosolic Ca2+ rise after ER Ca2+ store depletion with 2 μM Tg.

  4. Time‐course competitive ELISAs for estimating IP3 generation upon αMSH application (N = 3). Error bars indicate SD.

  5. Amplitude of αMSH‐induced ER Ca2+ release upon pre‐treatment of cells with PLC inhibitor, PLC inhibitor's inactive analog, and ADCY inhibitor.

  6. Forskolin induces rise in cytosolic Ca2+ in B16 cells in the absence of extracellular Ca2+.

Data information: Data presented in (E) are Mean ± SEM (***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.
Figure 6
Figure 6. STIM1 oligomerization regulates pigmentation and cAMP generation

  1. αMSH treatment induces sub‐plasmalemmal STIM1 oligomerization in B16 cells (please also refer to Movie EV1). Arrows point to sub‐plasmalemmal STIM1 puncta.

  2. Sub‐plasmalemmal intensity of STIM1 puncta measured in the same cell at resting state and upon αMSH stimulation (n = 40 cells).

  3. Cell pellets and melanin content analysis upon Tg treatment in B16 LD cultures and co‐treatment with 50 μM 2APB (N = 3).

  4. Cell pellets and melanin content analysis upon treatment with either vehicle control DMSO or αMSH alone or αMSH along with 2APB (N = 3).

  5. Melanin content analysis in the LD assay upon STIM1 silencing and its rescue with YFP‐hSTIM1 and SOAR mutant YFP‐hSTIM1 F394H (N = 6).

  6. ELISA‐based cAMP measurements upon Tg or TPEN treatment and upon co‐application of either 2APB or ML‐9 (N = 2).

  7. αMSH‐induced cAMP generation upon either STIM1 silencing or Orai1 knockdown in comparison with shLuciferase control (N = 2).

  8. cAMP generation upon STIM1 silencing and its rescue with YFP‐hSTIM1 and SOAR mutant YFP‐hSTIM1 F394H (N = 4).

Data information: Data represented are Mean ± SEM (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001; Student's t‐test).
Figure EV3
Figure EV3. STIM1 oligomerization regulates expression of key melanogenic genes
  1. A

    Confocal images of B16 cells transfected with eYFP‐STIM1 showing that Tg activates STIM1 oligomerization and 2APB disrupts STIM1 puncta. Arrows point to oligomeric STIM1 punta.

  2. B

    qRT–PCR data demonstrating that 2APB application for 48 h reduces mRNA expression of tyrosinase, TyRP1, and DCT (N = 3).

  3. C, D

    Gel images of tyrosinase activity and Western blots for DCT on LD day 7 treated with either vehicle control (DMSO) or 2‐APB.

  4. E

    qRT–PCR analysis showing that STIM1 silencing not only decreases mRNA expression of STIM1 but also that of tyrosinase, TyRP1, and DCT (N = 3).

  5. F

    qRT–PCR analysis showing that Orai1 knockdown decreases Orai1 mRNA levels, but expression of melanogenic genes remains largely unaffected (N = 3).

Data information: Data represented are Mean ± SEM (**P < 0.01, ***P < 0.001; unpaired Student's t‐test).
Figure 7
Figure 7. STIM1 physically interacts with ADCY6

  1. Melanin content analysis upon silencing of ADCYs in αMSH‐induced pigmentation and in LD melanogenesis assay and cell pellet pictures of the ADCY hits (N  = 2).

  2. The amplitude of αMSH‐induced Ca2+ release upon silencing of ADCY hits from αMSH screening along with an additional control of siADCY8.

  3. Blotting for ADCY6 upon immunoprecipitation of mCherry‐STIM1 with mCherry antibody demonstrating that STIM1 interacts with ADCY6 upon ER Ca2+ depletion.

  4. Blot showing reverse co‐IP wherein immunoprecipitation of ADCY6 was performed and subsequent blotting with STIM1 antibody.

  5. Scatter plots of AB FRET efficiencies demonstrate STIM1‐YFP and ADCY6‐CFP interact post‐ER Ca2+ depletion. Here, “n = x, y” represents x = number of ROIs and y = number of cells.

Data information: Data presented are Mean ± SEM (*P < 0.05; ***P < 0.001; unpaired Student's t‐test). The number of cells in (B) 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.
Figure EV4
Figure EV4. Constitutively active STIM1 rescues cAMP generation and melanogenesis

  1. Amplitude of αMSH‐induced Ca2+ release in WT cells and in cells overexpressing either wild‐type STIM1 (YFP‐STIM) or constitutively active STIM1 YFP‐STIM1 D76A.

  2. αMSH‐stimulated cAMP generation upon STIM1 silencing and its rescue with YFP‐STIM or YFP‐STIM1 D76A (N = 4).

  3. LD day 7 melanin content analysis in the B16 shSTIM1 stables and rescue with YFP‐STIM or YFP‐STIM1 D76A (N = 6).

  4. Melanin content analysis of B16 cells stimulated with αMSH in non‐transfected control cells and in cells overexpressing either YFP‐STIM1 D76A, ADCY6‐CFP, or YFP‐STIM1 D76A along with ADCY6‐CFP (N = 2).

Data information: Data represented are Mean ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student's t‐test). The number of cells in (A) 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.
Figure 8
Figure 8. STIM1 C‐terminus S/P‐rich domain regulates STIM1 interaction with ADCY6

  1. The cartoon representing the different hSTIM1 truncations employed in the study.

  2. Melanin content analysis in the LD melanogenesis assay performed with B16 shSTIM1 stables and STIM1 rescue with hSTIM1FL, STIM11–535, STIM11–485, STIM11–474, or STIM11–450 (N = 3).

  3. Amplitude of αMSH‐induced ER Ca2+ release in WT B16 cells and cell overexpression either STIM1‐YFP or STIM1 ΔS/P‐YFP.

  4. αMSH‐stimulated cAMP generation upon STIM1 silencing and its rescue with STIM1‐YFP, STIM1 ΔS/P‐YFP, or STIM1 ΔK‐YFP (N = 4).

  5. LD day 7 melanin content analysis in the B16 shSTIM1 stables and STIM1 rescue with STIM1‐YFP, STIM1 ΔS/P‐YFP, or STIM1 ΔK‐YFP (N = 6).

  6. Co‐IP blots showing that full‐length STIM1, but not STIM1 ?S/P, interacts with ADCY6 after ER Ca2+ depletion.

  7. Scatter plots of AB FRET efficiencies between STIM1 ΔS/P‐YFP and ADCY6‐CFP. Here, “n = x, y” represents x = number of ROIs and y = number of cells.

Data information: Data represented are Mean ± SD (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired Student's t‐test). The number of cells in (C) 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.
Figure EV5
Figure EV5. STIM1 C‐terminus S/P‐rich domain regulates STIM1‐mediated melanogenesis

  1. Domain architecture of hSTIM1 and the protein sequence analysis of C‐terminus of hSTIM1, mSTIM1, zSTIM1a, and zSTIM1b.

  2. Melanin content analysis in B16 shSTIM1 stables and its rescue with zSTIM1a and zSTIM1b (N = 5).

  3. Representative SOCE traces of B16 cells expressing either empty mCherry vector or mCherry‐zSTIM1b plasmid.

  4. The quantitative analysis of ER Ca2+ release in vector control and zSTIM1b‐expressing cells.

  5. The analysis of SOCE amplitude upon ectopic expression of zSTIM1b.

Data information: Data represented are Mean ± SEM (*P < 0.05; ***P < 0.001; unpaired Student's t‐test). (D, E) The number of cells are 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.
Figure 9
Figure 9. STIM1 activates Orai1 and ADCY6 at ER‐PM junctions for driving melanocyte proliferation and pigmentation
Schematic representation showing αMSH induces ER Ca2+ release and recruitment of STIM1 to ER‐PM junctions. STIM1 via its S/P domain interacts with ADCY6 at these junctions and regulates αMSH‐induced pigmentation. Additionally, STIM1 activates Orai1‐mediated Ca2+ influx for driving αMSH‐stimulated melanocyte proliferation. STIM1 therefore interacts with two distinct proteins at ER‐PM junctions for simultaneously driving two diverse cellular functions.
See this image and copyright information in PMC

Comment in

  • STIM1 (c)AMPs up melanogenesis.
    Soboloff J, Gligorijevic B, Zaidi MR. Soboloff J, et al. EMBO J. 2018 Mar 1;37(5):e99047. doi: 10.15252/embj.201899047. Epub 2018 Feb 15. EMBO J. 2018. PMID: 29449324 Free PMC article.

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