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TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus

TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).
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pone.0127480.g001: TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).

Mentions: Previously we described a high content microscopy based cDNA screen to identify genes that induced nuclear translocation of the CREB coactivator, CRTC1 [8]. This and other work demonstrated translocation of the CRTC1, is induced by elevations in cAMP or intracellular calcium through rapid calcineurin dependent dephosphorylation of CRTC1 [6,8]. In addition to known regulators of calcium and cAMP signaling, several novel proteins were identified and retested here for the ability to induce nuclear translocation of CRTC1. Of the genes analyzed, TMEM203 transfection resulted in efficient CRTC1 translocation without inducing gross morphologic and/or apoptotic changes (S1 Table). As shown in Fig 1A, exogenous expression of TMEM203 in CRTC1-eGFP expressing HeLa cells resulted in nuclear translocation which was blocked by the calcineurin inhibitors Cyclosporine A (CsA) and FK506. Addition of the calcium chelator, EGTA, also blocked TMEM203 induced nuclear CRTC1, and this inhibition was reversed by the addition of excess calcium (data not shown). In addition expression of TMEM203-FLAG induced nuclear localization of NFAT1(1–402)-GFP in cotransfected HeLa cells (Fig 1B). TMEM203 expression resulted in production of de-phosphorylated NFAT-GFP which was blocked by CsA or FK506 (Fig 1C).


TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).
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Related In: Results  -  Collection

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pone.0127480.g001: TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).
Mentions: Previously we described a high content microscopy based cDNA screen to identify genes that induced nuclear translocation of the CREB coactivator, CRTC1 [8]. This and other work demonstrated translocation of the CRTC1, is induced by elevations in cAMP or intracellular calcium through rapid calcineurin dependent dephosphorylation of CRTC1 [6,8]. In addition to known regulators of calcium and cAMP signaling, several novel proteins were identified and retested here for the ability to induce nuclear translocation of CRTC1. Of the genes analyzed, TMEM203 transfection resulted in efficient CRTC1 translocation without inducing gross morphologic and/or apoptotic changes (S1 Table). As shown in Fig 1A, exogenous expression of TMEM203 in CRTC1-eGFP expressing HeLa cells resulted in nuclear translocation which was blocked by the calcineurin inhibitors Cyclosporine A (CsA) and FK506. Addition of the calcium chelator, EGTA, also blocked TMEM203 induced nuclear CRTC1, and this inhibition was reversed by the addition of excess calcium (data not shown). In addition expression of TMEM203-FLAG induced nuclear localization of NFAT1(1–402)-GFP in cotransfected HeLa cells (Fig 1B). TMEM203 expression resulted in production of de-phosphorylated NFAT-GFP which was blocked by CsA or FK506 (Fig 1C).

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus