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Autocrine transforming growth factor β signaling regulates extracellular signal-regulated kinase 1/2 phosphorylation via modulation of protein phosphatase 2A expression in scleroderma fibroblasts.

Samuel GH, Bujor AM, Nakerakanti SS, Hant FN, Trojanowska M - Fibrogenesis Tissue Repair (2010)

Bottom Line: We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression.Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression.Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthritis Center, Division of Rheumatology, Boston University Medical Campus, Boston, MA, USA. trojanme@bu.edu.

ABSTRACT

Background: During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production.

Results: We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation.

Conclusions: Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.

No MeSH data available.


Related in: MedlinePlus

Increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation and decreased protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts. Dermal fibroblasts obtained from SSc patients and matched controls were grown to confluence and then serum starved for 24 h. Cells were collected and in (a) the levels of ERK1/2 phosphorylation were analyzed by western blot. In (b), the mRNA levels of the PP2A catalytic subunits α and β were analyzed using quantitative PCR. The mRNA values were normalized relative to matched controls (arbitrarily set as 1) and means ± standard error of the mean (SEM) of five independent experiments are shown (*P < 0.01). (c) The total protein levels of the PP2A catalytic subunit were measured by western blot. β Actin was used as a loading control.
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Figure 3: Increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation and decreased protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts. Dermal fibroblasts obtained from SSc patients and matched controls were grown to confluence and then serum starved for 24 h. Cells were collected and in (a) the levels of ERK1/2 phosphorylation were analyzed by western blot. In (b), the mRNA levels of the PP2A catalytic subunits α and β were analyzed using quantitative PCR. The mRNA values were normalized relative to matched controls (arbitrarily set as 1) and means ± standard error of the mean (SEM) of five independent experiments are shown (*P < 0.01). (c) The total protein levels of the PP2A catalytic subunit were measured by western blot. β Actin was used as a loading control.

Mentions: To further study the relationship of PP2A and ERK1/2 phosphorylation in the pathological context, age, race and gender matched SSc and normal dermal fibroblasts obtained from patient biopsy were analyzed for PP2A expression and ERK1/2 activation. ERK1/2 phosphorylation was increased in SSc fibroblasts, consistent with data from previous reports [12] (Figure 3a). The mRNA levels of both isoforms of the PP2A C-subunit were significantly decreased in SSc fibroblasts when compared to normal controls (Figure 3b). Consistent with the mRNA data, the protein levels of the catalytic subunit of PP2A were significantly lower in SSc fibroblasts compared to normal controls (Figure 3c). The observations from SSc fibroblasts are consistent with the results from normal fibroblasts treated with TGFβ that show PP2A downregulation, suggesting a role for TGFβ in mediating these changes in SSc fibroblasts.


Autocrine transforming growth factor β signaling regulates extracellular signal-regulated kinase 1/2 phosphorylation via modulation of protein phosphatase 2A expression in scleroderma fibroblasts.

Samuel GH, Bujor AM, Nakerakanti SS, Hant FN, Trojanowska M - Fibrogenesis Tissue Repair (2010)

Increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation and decreased protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts. Dermal fibroblasts obtained from SSc patients and matched controls were grown to confluence and then serum starved for 24 h. Cells were collected and in (a) the levels of ERK1/2 phosphorylation were analyzed by western blot. In (b), the mRNA levels of the PP2A catalytic subunits α and β were analyzed using quantitative PCR. The mRNA values were normalized relative to matched controls (arbitrarily set as 1) and means ± standard error of the mean (SEM) of five independent experiments are shown (*P < 0.01). (c) The total protein levels of the PP2A catalytic subunit were measured by western blot. β Actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008687&req=5

Figure 3: Increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation and decreased protein phosphatase 2A (PP2A) expression in scleroderma (SSc) fibroblasts. Dermal fibroblasts obtained from SSc patients and matched controls were grown to confluence and then serum starved for 24 h. Cells were collected and in (a) the levels of ERK1/2 phosphorylation were analyzed by western blot. In (b), the mRNA levels of the PP2A catalytic subunits α and β were analyzed using quantitative PCR. The mRNA values were normalized relative to matched controls (arbitrarily set as 1) and means ± standard error of the mean (SEM) of five independent experiments are shown (*P < 0.01). (c) The total protein levels of the PP2A catalytic subunit were measured by western blot. β Actin was used as a loading control.
Mentions: To further study the relationship of PP2A and ERK1/2 phosphorylation in the pathological context, age, race and gender matched SSc and normal dermal fibroblasts obtained from patient biopsy were analyzed for PP2A expression and ERK1/2 activation. ERK1/2 phosphorylation was increased in SSc fibroblasts, consistent with data from previous reports [12] (Figure 3a). The mRNA levels of both isoforms of the PP2A C-subunit were significantly decreased in SSc fibroblasts when compared to normal controls (Figure 3b). Consistent with the mRNA data, the protein levels of the catalytic subunit of PP2A were significantly lower in SSc fibroblasts compared to normal controls (Figure 3c). The observations from SSc fibroblasts are consistent with the results from normal fibroblasts treated with TGFβ that show PP2A downregulation, suggesting a role for TGFβ in mediating these changes in SSc fibroblasts.

Bottom Line: We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression.Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression.Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthritis Center, Division of Rheumatology, Boston University Medical Campus, Boston, MA, USA. trojanme@bu.edu.

ABSTRACT

Background: During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production.

Results: We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation.

Conclusions: Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.

No MeSH data available.


Related in: MedlinePlus