Poster Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Regulation of activin A production and fibrotic transformation by cultured human lung fibroblasts and the effects of blocking activin action by follistatin (#224)

Adnan Sali 1 , David M de Kretser 1 , Mark P Hedger 1
  1. MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia

Transforming growth factor-β (TGFβ) and activin A increase in lung inflammation. Follistatin, which binds activin A, but not TGFβ, modulates post-inflammation fibrosis in the lung in vivo. This implies a crucial role for activin A in lung fibrosis, which was investigated at the cellular level in adult human lung fibroblasts (Lonza). When maintained in fibroblast growth medium (FGMTM) containing basic fibroblast growth factor (bFGF) and 2% FCS, these cells express low levels of the fibrosis genes, collagen 1A1 (Col1A1) and α-smooth muscle (αSMA), measured by qRT-PCR. For experiment, cells were first expanded in 10% FCS, without bFGF, then seeded into 6 well plates at low density and incubated for 4h. After attachment, the cells were washed with PBS and transferred to serum-free medium. Under these conditions, the cells produced both activin A, measured by ELISA, and follistatin, measured by RIA. Production of activin A was stimulated by TGFβ1 and by rh-thrombin, known inducers of fibrosis, although not by angiotensin II or endothelin-1. Significantly, bFGF had no effect on activin A production, but the effects of TGFβ1 were potentiated by bFGF. Transfer to low cell density under serum-free conditions led to increased expression of Col1A1 and αsm-actin, and this transition was accelerated by TGFβ1. Addition of 300 ng/ml rh-follistatin 288 reduced activin A levels in culture and reduced expression of Col1A1 and αSMA in the presence of TGFβ1. These data confirm that cultured adult lung fibroblasts produce activin A and undergo fibrotic differentiation in the absence of bFGF, a process that is stimulated by TGFβ1. Blocking activin A action inhibits this process, highlighting a facilitative role for activin A in both spontaneous fibrotic differentiation and in mediating the pro-fibrotic effects of TGFβ1. The data also suggest that bFGF reduces the transition to fibrosis by blocking pro-fibrotic actions downstream of TGFβ1/activin A.