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Prostaglandin F2(alpha) stimulates growth of skeletal muscle cells via an NFATC2-dependent pathway.

Horsley V, Pavlath GK - J. Cell Biol. (2003)

Bottom Line: We demonstrate that prostaglandin F2alpha (PGF2alpha) as well as two analogues augment muscle cell size in vitro.We show that NFAT is activated by PGF2alpha, and the isoform NFATC2 is required for PGF2alpha-induced muscle cell growth and nuclear accretion, demonstrating the first intersection between prostaglandin receptor activation and NFAT signaling.Given this novel role for PGF2alpha in skeletal muscle cell growth, these studies raise caution that extended use of drugs that inhibit PG production, such as nonsteroidal antiinflammatory drugs, may be deleterious for muscle growth.

View Article: PubMed Central - PubMed

Affiliation: Cell and Developmental Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Skeletal muscle growth requires multiple steps to form large multinucleated muscle cells. Molecules that stimulate muscle growth may be therapeutic for muscle loss associated with aging, injury, or disease. However, few factors are known to increase muscle cell size. We demonstrate that prostaglandin F2alpha (PGF2alpha) as well as two analogues augment muscle cell size in vitro. This increased myotube size is not due to PGF2alpha-enhancing cell fusion that initially forms myotubes, but rather to PGF2alpha recruiting the fusion of cells with preexisting multinucleated cells. This growth is mediated through the PGF2alpha receptor (FP receptor). As the FP receptor can increase levels of intracellular calcium, the involvement of the calcium-regulated transcription factor nuclear factor of activated T cells (NFAT) in mediating PGF2alpha-enhanced cell growth was examined. We show that NFAT is activated by PGF2alpha, and the isoform NFATC2 is required for PGF2alpha-induced muscle cell growth and nuclear accretion, demonstrating the first intersection between prostaglandin receptor activation and NFAT signaling. Given this novel role for PGF2alpha in skeletal muscle cell growth, these studies raise caution that extended use of drugs that inhibit PG production, such as nonsteroidal antiinflammatory drugs, may be deleterious for muscle growth.

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PGF2α does not induce myotube growth or fusion of NFATC2−/− muscle cells. (A) NFATC2−/− myoblasts were induced to differentiate and treated with vehicle or 10−6 M PGF2α for 48 h, followed by immunostaining for EMyHC. Bar, 60 μm. (B) Nuclear number assays were performed as in Fig. 1 E on NFATC2−/− cultures treated with vehicle or PGF2α at a range of doses. Data are the mean ± SEM of three independent cell isolates. (C) FP receptor mRNA expression was examined by RT-PCR after 24 h in DM in wild-type and NFATC2−/− muscle cells. Representative ethidium bromide staining of acrylamide gel is shown with 18S rRNA as an internal control.
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fig5: PGF2α does not induce myotube growth or fusion of NFATC2−/− muscle cells. (A) NFATC2−/− myoblasts were induced to differentiate and treated with vehicle or 10−6 M PGF2α for 48 h, followed by immunostaining for EMyHC. Bar, 60 μm. (B) Nuclear number assays were performed as in Fig. 1 E on NFATC2−/− cultures treated with vehicle or PGF2α at a range of doses. Data are the mean ± SEM of three independent cell isolates. (C) FP receptor mRNA expression was examined by RT-PCR after 24 h in DM in wild-type and NFATC2−/− muscle cells. Representative ethidium bromide staining of acrylamide gel is shown with 18S rRNA as an internal control.

Mentions: Given the requirement of NFAT in PGF2α-induced muscle growth, direct activation of NFAT signaling by PGF2α was analyzed in muscle cells. First, cells were transiently transfected with an NFATC2-GFP fusion construct and treated with 10−6 M PGF2α after 24 h in DM. Without treatment, cells expressing NFATC2-GFP exhibit GFP throughout the cell (Fig. 4 A). Stimulation with PGF2α results in translocation and accumulation of NFAT in nuclei of myotubes. This nuclear translocation of NFAT is blocked by treatment with cyclosporine A (CsA), suggesting that calcineurin is activated by PGF2α. In addition, PGF2α-induced nuclear translocation of NFATC2 is inhibited by cotreatment with the FP antagonist, AL-8810. Thus, stimulation of the FP receptor is responsible for NFAT activation and not a nonspecific increase in intracellular calcium at this dose of PGF2α. Transcriptional activity of NFAT was analyzed also in cells containing a NFAT responsive luciferase reporter construct (Fig. 5 B). Luciferase activity is increased in cells treated with 10−6 M PGF by approximately ninefold. Together, these data indicate that PGF2α activates NFAT signaling in skeletal muscle.


Prostaglandin F2(alpha) stimulates growth of skeletal muscle cells via an NFATC2-dependent pathway.

Horsley V, Pavlath GK - J. Cell Biol. (2003)

PGF2α does not induce myotube growth or fusion of NFATC2−/− muscle cells. (A) NFATC2−/− myoblasts were induced to differentiate and treated with vehicle or 10−6 M PGF2α for 48 h, followed by immunostaining for EMyHC. Bar, 60 μm. (B) Nuclear number assays were performed as in Fig. 1 E on NFATC2−/− cultures treated with vehicle or PGF2α at a range of doses. Data are the mean ± SEM of three independent cell isolates. (C) FP receptor mRNA expression was examined by RT-PCR after 24 h in DM in wild-type and NFATC2−/− muscle cells. Representative ethidium bromide staining of acrylamide gel is shown with 18S rRNA as an internal control.
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Related In: Results  -  Collection

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fig5: PGF2α does not induce myotube growth or fusion of NFATC2−/− muscle cells. (A) NFATC2−/− myoblasts were induced to differentiate and treated with vehicle or 10−6 M PGF2α for 48 h, followed by immunostaining for EMyHC. Bar, 60 μm. (B) Nuclear number assays were performed as in Fig. 1 E on NFATC2−/− cultures treated with vehicle or PGF2α at a range of doses. Data are the mean ± SEM of three independent cell isolates. (C) FP receptor mRNA expression was examined by RT-PCR after 24 h in DM in wild-type and NFATC2−/− muscle cells. Representative ethidium bromide staining of acrylamide gel is shown with 18S rRNA as an internal control.
Mentions: Given the requirement of NFAT in PGF2α-induced muscle growth, direct activation of NFAT signaling by PGF2α was analyzed in muscle cells. First, cells were transiently transfected with an NFATC2-GFP fusion construct and treated with 10−6 M PGF2α after 24 h in DM. Without treatment, cells expressing NFATC2-GFP exhibit GFP throughout the cell (Fig. 4 A). Stimulation with PGF2α results in translocation and accumulation of NFAT in nuclei of myotubes. This nuclear translocation of NFAT is blocked by treatment with cyclosporine A (CsA), suggesting that calcineurin is activated by PGF2α. In addition, PGF2α-induced nuclear translocation of NFATC2 is inhibited by cotreatment with the FP antagonist, AL-8810. Thus, stimulation of the FP receptor is responsible for NFAT activation and not a nonspecific increase in intracellular calcium at this dose of PGF2α. Transcriptional activity of NFAT was analyzed also in cells containing a NFAT responsive luciferase reporter construct (Fig. 5 B). Luciferase activity is increased in cells treated with 10−6 M PGF by approximately ninefold. Together, these data indicate that PGF2α activates NFAT signaling in skeletal muscle.

Bottom Line: We demonstrate that prostaglandin F2alpha (PGF2alpha) as well as two analogues augment muscle cell size in vitro.We show that NFAT is activated by PGF2alpha, and the isoform NFATC2 is required for PGF2alpha-induced muscle cell growth and nuclear accretion, demonstrating the first intersection between prostaglandin receptor activation and NFAT signaling.Given this novel role for PGF2alpha in skeletal muscle cell growth, these studies raise caution that extended use of drugs that inhibit PG production, such as nonsteroidal antiinflammatory drugs, may be deleterious for muscle growth.

View Article: PubMed Central - PubMed

Affiliation: Cell and Developmental Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Skeletal muscle growth requires multiple steps to form large multinucleated muscle cells. Molecules that stimulate muscle growth may be therapeutic for muscle loss associated with aging, injury, or disease. However, few factors are known to increase muscle cell size. We demonstrate that prostaglandin F2alpha (PGF2alpha) as well as two analogues augment muscle cell size in vitro. This increased myotube size is not due to PGF2alpha-enhancing cell fusion that initially forms myotubes, but rather to PGF2alpha recruiting the fusion of cells with preexisting multinucleated cells. This growth is mediated through the PGF2alpha receptor (FP receptor). As the FP receptor can increase levels of intracellular calcium, the involvement of the calcium-regulated transcription factor nuclear factor of activated T cells (NFAT) in mediating PGF2alpha-enhanced cell growth was examined. We show that NFAT is activated by PGF2alpha, and the isoform NFATC2 is required for PGF2alpha-induced muscle cell growth and nuclear accretion, demonstrating the first intersection between prostaglandin receptor activation and NFAT signaling. Given this novel role for PGF2alpha in skeletal muscle cell growth, these studies raise caution that extended use of drugs that inhibit PG production, such as nonsteroidal antiinflammatory drugs, may be deleterious for muscle growth.

Show MeSH
Related in: MedlinePlus