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Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis.

Cencetti F, Bernacchioni C, Nincheri P, Donati C, Bruni P - Mol. Biol. Cell (2010)

Bottom Line: Furthermore, downstream of S1P(3), Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFbeta1.Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P(2), consequently to TGFbeta1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action.Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, 50134, Firenze, Italy.

ABSTRACT
The pleiotropic cytokine transforming growth factor (TGF)-beta1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFbeta1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFbeta1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFbeta1. Moreover, inhibition of S1P(3), which became the highest expressed S1PR after TGFbeta1 challenge, strongly attenuated the profibrotic response to TGFbeta1. Furthermore, downstream of S1P(3), Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFbeta1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P(2), consequently to TGFbeta1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFbeta1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

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TGFβ1 affects sphingolipid metabolite levels in C2C12 myoblasts. C2C12 myoblasts were labeled for 48 h with [3-3H]sphingosine. Cellular lipids were extracted, separated, and analyzed as described in Materials and Methods section. Data represent the mean ± SEM of three independent experiments.
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Figure 2: TGFβ1 affects sphingolipid metabolite levels in C2C12 myoblasts. C2C12 myoblasts were labeled for 48 h with [3-3H]sphingosine. Cellular lipids were extracted, separated, and analyzed as described in Materials and Methods section. Data represent the mean ± SEM of three independent experiments.

Mentions: To further examine the effect of TGFβ1 on sphingolipid metabolism, myoblasts, previously steady-state metabolically labeled with [3H]sphingosine, were administered with the cytokine (5 ng/ml) for various time intervals and then subjected to lipid extraction and HPTLC separation followed by digital autoradiography to quantify the various labeled sphingolipid metabolites. Data presented in Figure 2 show that [3H]ceramide levels were enhanced by TGFβ1 within 6 h, whereas at more prolonged times (18–24 h) they were significantly reduced. In parallel, [3H]sphingosine levels where not appreciably modified throughout the incubation. Interestingly, treatment with TGFβ1 at 18 and 24 h raised [3H]S1P levels by ∼60%. Thus, consistently with the complex regulation of SK activity, TGFβ1 elicited changes of cellular bioactive sphingolipid metabolite levels.


Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis.

Cencetti F, Bernacchioni C, Nincheri P, Donati C, Bruni P - Mol. Biol. Cell (2010)

TGFβ1 affects sphingolipid metabolite levels in C2C12 myoblasts. C2C12 myoblasts were labeled for 48 h with [3-3H]sphingosine. Cellular lipids were extracted, separated, and analyzed as described in Materials and Methods section. Data represent the mean ± SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836962&req=5

Figure 2: TGFβ1 affects sphingolipid metabolite levels in C2C12 myoblasts. C2C12 myoblasts were labeled for 48 h with [3-3H]sphingosine. Cellular lipids were extracted, separated, and analyzed as described in Materials and Methods section. Data represent the mean ± SEM of three independent experiments.
Mentions: To further examine the effect of TGFβ1 on sphingolipid metabolism, myoblasts, previously steady-state metabolically labeled with [3H]sphingosine, were administered with the cytokine (5 ng/ml) for various time intervals and then subjected to lipid extraction and HPTLC separation followed by digital autoradiography to quantify the various labeled sphingolipid metabolites. Data presented in Figure 2 show that [3H]ceramide levels were enhanced by TGFβ1 within 6 h, whereas at more prolonged times (18–24 h) they were significantly reduced. In parallel, [3H]sphingosine levels where not appreciably modified throughout the incubation. Interestingly, treatment with TGFβ1 at 18 and 24 h raised [3H]S1P levels by ∼60%. Thus, consistently with the complex regulation of SK activity, TGFβ1 elicited changes of cellular bioactive sphingolipid metabolite levels.

Bottom Line: Furthermore, downstream of S1P(3), Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFbeta1.Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P(2), consequently to TGFbeta1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action.Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, 50134, Firenze, Italy.

ABSTRACT
The pleiotropic cytokine transforming growth factor (TGF)-beta1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFbeta1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFbeta1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFbeta1. Moreover, inhibition of S1P(3), which became the highest expressed S1PR after TGFbeta1 challenge, strongly attenuated the profibrotic response to TGFbeta1. Furthermore, downstream of S1P(3), Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFbeta1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P(2), consequently to TGFbeta1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFbeta1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

Show MeSH
Related in: MedlinePlus