A reduction in Homeodomain Interacting Protein Kinase-2 in lung fibroblasts ameliorates lung fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive, and scarring lung disease with an unknown etiology. Homeodomain Interacting Protein Kinase 2 ( HIPK2) is a co-transcriptional regulator involved in kidney fibrosis. We hypothesize that a decrease in HIPK2 expression attenuates the activation and proliferation of lung fibroblasts through inhibition of TGF-β1 and Wnt 3a pathways. HIPK2 knockdown using a lentiviral shRNA in human pulmonary fibroblasts (HPFs) reduced TGF-β1- and WNT3a-induced expression of fibrotic markers including α-smooth actin, fibronectin, and collagen 1A1 and 3A1 as determined by Western blots and real-time PCR. Immunostaining of α-smooth actin showed that HIPK2 knockdown inhibited fiber formation in HPFs. Furthermore, HIPK2 knockdown suppressed fibroblast proliferation. HIPK2 knockdown in HPFs also resulted in a reduction in TGF-β1-induced SMAD 2/3 phosphorylation and WNT3a-induced activated β-Catenin expression, suggesting the involvement of HIPK2 regulation on above pathways. To investigate the effects of HIPK2 knockdown on lung fibrosis in mice, HIPK2 was knocked down (KD) in the lungs of mice using an adenovirus expressing 4 shRNAs targeting HIPK2 and lung fibrosis was induced by an intratracheal instillation of bleomycin (1U/kg body weight) for 14 days. HIPK2 KD mice lost less body weight compared to the control mice. Analysis of bronchoalveolar lavage (BAL) indicated a significant reduction in total infiltrated immune cells in Hipk2 KD mice. Lung fibrosis was reduced in Hipk2 KD mice as demonstrated by fibrotic score, Sirius red staining, fibronectin mRNA level, and collagen content as measured by hydroxyproline assay. Lung functions including elastance, compliance, resistance, and inspiratory capacity, as determined by Flexivent, were improved in Hipk2 KD mice. Real-time PCR and ELISA revealed a significantly reduced mRNA expression of Tgf-β1 in the lung tissues and a decreased TGF-β1 level in BAL fluids of Hipk2 KD mice. Immunofluorescence indicated reduced SMAD-3 and activated β-Catenin levels in the fibrotic regions of Hipk2 KD mice. These findings clearly suggest that reduced HIPK2 expression in the lung attenuates lung fibrosis through the suppression of TGF-β1 and WNT3a signaling pathways. R01 HL157450, R01HL135152 and P20GM103648 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.