Role of CARM1 in regulation of alveolar epithelial senescence and emphysema susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases world-wide. A major feature of COPD is emphysema- a long-term, progressive condition. The hallmark of emphysema includes the destruction of alveolar structures leading to enlarged air spaces and reduced surface area. Experimental evidence suggests that emphysema development is driven by accelerated senescence of lung cells but the underlying mechanism of senescence is yet to be fully elucidated. Protein arginine methyltransferases (PRMTs) are important for cellular processes, such as the regulation of senescence, cell proliferation, differentiation and apoptosis. The PRMT family includes 11 members classified as type I, II or III enzymes depending on their methylation pattern (asymmetric dimethylation, symmetric dimethylation or monomethylation, respectively). One member of this family is PRMT4, a type I enzyme, which is also called coactivator associated arginine methyltransferase 1 (CARM1). It was originally identified as a coactivator for steroid hormone receptors. CARM1 is known to methylate histone H3 and various non-histone proteins that play essential roles in transcriptional regulation, RNA splicing, and metabolism. Most importantly, complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type-II cells (ATII). Furthermore, CARM1 also plays a role in regulating cellular senescence via CARM1-dependent methylation. Based on these reports, we hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and siRNA-mediated knockdown in ATII-like LA4 cells in vitro. We monitored emphysema progression for 161 days in mice treated with a single oropharyngeal application of elastase. The progression was manifested by the decline in lung function parameters. The mean chord length (Lm) confirmed a time dependent airspace enlargement and was directly correlated with a significant increase in dynamic lung compliance. We also observed that at later time points (day 56 and 161), emphysema progression was inflammation-independent. We demonstrated that emphysema advancement was associated with a time-dependent downregulation of CARM1, specifically in alveolar epithelial cells. Furthermore, the global CARM1 activity was also reduced as reflected by an elevated level of CARM1 phosphorylation in the lung. Most importantly, elastase-treated CARM1 haploinsufficient mice showed significantly increased airspace enlargement (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) and lung compliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) compared with wild type controls. Reduced CARM1 contributed to senescence of alveolar epithelial cells evident by the reduction of anti-senescence SIRT1 and the induction of senescence markers p16 and β-galactosidase in alveolar epithelial cells. We further demonstrated that CARM1 haploinsufficiency impaired trans-differentiation of ATII into ATI cells. Elastase treatment in CARM1 deficient mouse lungs led to the accumulation of SP-C positive ATII cell. In our in vitro studies, we detected that the knockdown of CARM1 in the ATII-like cell line LA-4 led to decreased SIRT1 expression (0.034±0.003 vs. 0.022±0.001, p<0.05), but increased expression of p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) and induced a higher number of β-galactosidase-positive senescent cells (50.57%±7.36 vs. 2.21%±0.34, p<0.001), compared with scrambled siRNA. The senescence in CARM1 deficient cells was further augmented after CSE stimulation. Furthermore, we demonstrated that CARM1 deficiency impaired wound healing (32.18%±0.9512 vs. 8.769%±1.967 wound gap closure, p<0.001) of alveolar epithelial cells. The data confirmed that CARM1 reduction induced an accelerated senescence in LA-4 cells by attenuating the effect of SIRT1. Overall, both our in vivo and in vitro results revealed a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence, as well as impaired regeneration, repair and differentiation of ATII cells