Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease characterized by excessive scarring and fibroblast activation. We previously showed that fibroblasts from patients with IPF are hypermethylated at the CDKN2B gene locus, resulting in decreased CDKN2B expression. Here, we examine how diminished CDKN2B expression in normal and IPF fibroblasts affect fibroblast function, and how loss of CDKN2B contributes to IPF pathogenesis. We first confirmed that protein expression of CDKN2B was diminished in IPF lungs in situ. Loss of CDKN2B was especially notable in regions of increased myofibroblasts and fibroblastic foci. The degree of CDKN2B hypermethylation was particularly elevated in patients with radiographic honeycombing, a marker of more advanced fibrosis, and increased DNA methylation correlated with decreased expression. Although CDKN2B is traditionally considered a cell cycle inhibitor, loss of CDKN2B did not result in an increase in fibroblast proliferation, but instead was associated with an increase in myofibroblast differentiation. An increase in myofibroblast differentiation was not observed when CDKN2A was silenced. Loss of CDKN2B was associated with an increase in the transcription factors serum response factor and myocardin-related transcription factor A, and overexpression of CDKN2B in IPF fibroblasts inhibited myofibroblast differentiation. Finally, decreased CDKN2B expression was noted in fibroblasts from a murine model of fibrosis, and Cdkn2b-/- mice developed greater histologic fibrosis after bleomycin injury. These findings identify a novel function for CDKN2B that differs from its conventional designation as a cell cycle inhibitor and demonstrate the importance of this protein in pulmonary fibrosis.

Transforming Growth Factor-ß1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts.

DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-ß1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-ß1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-ß1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-ß1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-ß1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-ß1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-ß1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3ß, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3ß was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-ß1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-ß1 is known to be crucial, the ability of TGF-ß1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-ß1 may regulate DNA methylation in these cells.