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Reversal of TGF-?1 stimulation of ?-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts.

ABSTRACT: BACKGROUND:Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (?-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-?1)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis. METHODS:Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-?1 (2 ng/ml) and/or forskolin (10 ?M) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis. RESULTS:The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-?1 stimulation of ?-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-?1 stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types. CONCLUSION:In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.

SUBMITTER: Satish L

PROVIDER: S-EPMC3125251 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts.

Satish Latha L Gallo Phillip H PH Baratz Mark E ME Johnson Sandra S Kathju Sandeep S

BMC musculoskeletal disorders 20110525

<h4>Background</h4>Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP ( ...[more]

PMID: 21612641

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Project description:Cellular functions are regulated by complex networks of many different signaling pathways. The TGF? and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGF?-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and T?RI (TGF? receptor 1), were responsive to cAMP. While YAP had little effect on TGF?-dependent expression and Smad3 phosphorylation, a constitutively active form of T?RI mimicked the cAMP effect on TGF? signaling. In 3D-cultured cells, which show much higher levels of T?RI and cAMP, T?RI was unresponsive to cAMP. Upregulation of T?RI expression by cAMP was dependent on transcription. A proximal T?RI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases T?RI expression at least partially by activating T?RI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the T?RI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased T?RI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGF? on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGF? pathways. In summary, these data suggest that combined effects of cAMP and TGF?, as e.g. induced by mesenchymal stem cells, involve the upregulation of T?RI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

Project description:Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100 mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5 mM 2-O-α-d-glucopyranosyl-l-ascorbic acid (VitC) ± 2 ng/ml of T1 or T3. After 4 h or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 h, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

Dataset Information

Reversal of TGF-?1 stimulation of ?-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts.

Publications

Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts.

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