Blocking the Phase Separation of hnRNP L Attenuates Pulmonary Fibrogenesis by Inhibiting Fibroblast-to-myofibroblast Differentiation.

Pulmonary fibrosis is an interstitial lung disease characterized by diverse etiologies and unknown mechanisms. Phase separation has been implicated in the development of numerous diseases, but its role in the pathogenesis of pulmonary fibrogenesis remains unclear. In this study, we explored whether heterogeneous nuclear ribonucleoprotein L (hnRNP L) can form phase-separation condensates that contribute to the progression of pulmonary fibrosis. Pulmonary fibrosis was modeled in vitro using TGF-β1-treated fibroblasts and in vivo by intratracheal bleomycin administration in mice. Phase-separation formation was evaluated by droplet formation and fluorescence recovery after photobleaching experiments. Adeno-associated virus vectors overexpressing full-length hnRNP L or hnRNP L-Δ26-111 & 274-374 (truncation of amino acid fragments 26-111 and 274-374) were constructed and delivered into murine lungs using a Penn-Century MicroSprayer device. Micro-computed tomography imaging, forced vital capacity tests, hematoxylin and eosin staining, and Masson's trichrome staining were performed to assess the anti-fibrotic effect of disrupting hnRNP L phase separation. RNA-seq was employed to explore the underlying molecular targets of hnRNP L. In vivo and in vitro results revealed that the role of hnRNP L in promoting fibroblast differentiation depended on its phase separation, and the amino acid fragments 26-111 and 274-374 were essential to this process. Phenotypic, physiological, and histological analyses demonstrated that hnRNP L overexpression accelerated the development of pulmonary fibrogenesis. In contrast, truncated hnRNP L-Δ26-111 and 274-374 improved lung tissue structure and function and reduced collagen deposition by blocking hnRNP L phase separation. RNA-seq results showed that a total of 1,155 dysregulated genes (FDR < 0.05, |logFC| > 1) were detected after silencing hnRNP L using small interfering RNA (siRNA) in TGF-β1-treated MRC-5 cells. Most differentially expressed genes were related to lung fibrosis, including TGFBR1, CXCL8, WWTR1, FAP, VIM, α-SMA, and collagen. The RNA levels of these genes were evidently regulated by silencing or overexpressing hnRNP L. Notably, we observed that hnRNP L phase separation promoted histone modifications such as H3K27ac, H4K8la, H4K12la, and H4K16la, which are associated with active chromatin. Our findings reveal that TGF-β1-enhanced phase separation of the chromatin-associated RBP hnRNP L promotes gene transcription and facilitates profibrotic phenotypes in lung myofibroblasts, suggesting a phase-separation-based therapeutic strategy. Blocking hnRNP L phase separation inhibited fibroblast-to-myofibroblast differentiation, attenuating pulmonary fibrogenesis. This study reveals a novel regulatory mechanism by which phase separation contributes to the development of pulmonary fibrosis.