The Role Of HMGB1 In Pulmonary Fibrosis

Rationale:Idiopathic pulmonary fibrosis (IPF), which is the most common idiopathic interstitial lung disease, has the worse prognosis, with a median survival of only 3 to 4 years. Although much has been learned recently about the pathogenesis of IPF, the etiology and precise cellular and molecular mechanisms involved are not established. So far, no specific therapy has been proven unequivocally effective in IPF. The most notable characteristic feature of IPF is presence of fibroblastic foci. Increased numbers of fibroblastic foci are associated with disease progression and a worse prognosis in IPF. The key effector cell in fibroblastic foci is the myofibroblast. Myofibroblasts, with their characteristicα-smooth muscle actin (α-SMA) expression, arise de novo in fibrosis, and they are thought to be the primary source of heightened matrix and profibrogenic cytokine expression. Hence, understanding the mechanism responsible for abnormal activation and transcriptional regulation ofα-SMA gene is important to uncover the fibrotic pathogenesis. We have previously identified a 20Kda nuclear protein (GenBank number:BAC34367) that is a C-terminal truncated form of high mobility group B-1(HMGB1) in the lung of bleomycin-treated mice, with an increased binding toα-SMA promoter. HMGB1 is a nuclear protein that is present in almost all eukaryotic cells, and it functions to stabilize nucleosome formation and acts as a transcription-factor like protein that regulates the expression of several genes. Recent studies identify HMGB1 as a delayed mediator of inflammation, damaged or necrotic cells can release HMGB1 into the extracellular milieu, where it triggers inflammatory responses. Based on these findings described as above, we hypothesize that HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF. To validate the contribution of HMGB1 to the pathogenesis of the disease, we evaluated the role of HMGB34367, a C-terminal truncated form of HMGB1, in the initiation and progression of lung fibrosis induced by BLM, especially clarified its role in epithelial-myofibroblast transdifferentiation underlying the pathogenesis of IPF.MethodsThe first part:Investigation of the expression of HMGB1 on the lung tissues of mice with pulmonary fibrosis induced by BLM and IPF patients.The lung tissues sections were prepared from BLM-treated mice and IPF patients according to routine protocols for pathological examination, where immunostainings toα-SMA and HMGB1 were performed on determination of the role of HMGB1 in the lung fibrotic process.The second part:Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosis To validate if the truncated form of HMGB1 is involved in activation of myofibroblasts, the BLM-treated mice were administered intranasally with either HMGB34367 siRNA or control siRNA (20μl /per time )on day 2, 5, 8 after BLM addition. The lung tissues were isolated for pathological examination. Immunostaining toα-SMA and HMGB1 and western blot were performed for determination of effect of the increased level of HMGB1 on activation of myofibroblasts.The third part:The role of HMGB1 in the transdifferentiation of pulmonary epithelial cells into myofibroblastsThe 16HBE cells transfected with pcDNA3.0-HMGB34367 were subjected to performance of Electrophoretic Mobility Shift Assay(EMSA) and Super shift for detection of the binding activity of HMGB34367 withα-SMA promoter CarGB motif. The flowcytometry assay for measurement of the ratio of red fluorescence (RFP protein expressed under guidance ofα-SMA promoter) to green fluorescence of GPF-HMGB34367 fusion protein .was performed on qualification ofα-SMA promoter's activation in response to over-expression of HMGB34367 in the cells after co-transfected with pGPF-HMGB34367 and pDsRed-SMA . To elucidate endogenous induction of HMGB1 by inflammatory micro-environment in lung fibrotic process,the A549 cells were cultured with cytokines-mix (TGF-β1+TNF-α+ IFN-γ) in absence or presence of HMGB34367-RNAi followed by RT-PCR and immunofluorescent staining to test expression ofα-SMA and HMGB1,ResultThe first part:Investigation of the expression of HMGB1 on the lung tissues of BLM-induced pulmonary fibrosis mice and IPF.Pathological examination demonstrated that BLM administration induced focal fibrotic lesions in mice lungs, primarily in the subpleural regions with thickened or thickening interalveolar septa with obvious alveolar destruction, mesenchyma infiltration with inflammatory cells and epithelial shedding. By Masson's trichrome staining, it was demonstrated an increased level of collagen synthesis. .Immunostaining displayed expression of both HMGB1 andα-SMA with a significant elevated level on the lung tissues of BLM-treated mice. In the pulmonary tissue of patients with IPF, we observed the same as above. Using double immunofluorescent staining, with antibodies againstα-SMA and HMGB1, we further observed certain cells located at bronchiolar epithelium of the pulmonary tissue of IPF with double staining ofα-SMA and HMGB1.The second part:Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosisTreatment of HMGB34367 siRNA significantly attenuated the lung fibrotic lesion in the mice induced by BLM. Correspondingly, immunohistochemistry examination demonstrated that compare with the mice with challenge by BLM alone or plus with siRNA control, the expression of HMGB1 were obviously decreased in the BLM-induced mice with the treatment of HMGB34367 siRNA .,α-SMA expression in the injured epithelial cells and subepithelial cells was correspondingly down-regulated .The same results were also detected by Western-Blot.The third part:The role of HMGB1 in the transdifferentiation of airway epithelial cells into myofibroblastsEMSA and super shif revealed that the truncated form of HMGB1 can specifically bind to CArG B motif. By Flow cytometry, without and with TGFβ1, RFP/GFP signal ratio was detected with a higher level in the cells co-transfected with pDsRed-SMA+pEGFP-N2-HMGB34367 plasmids than those co-transfected with control plasmids. Following with treatment of cytokines-mix, the A549 cells appeared myofibroblastic morphology concomitant with highly expressed intracellular HMGB1. As shown by the results of RT-PCR and immunofluorescent staining, transcriptional induction ofα-SMA and HMGB34367 genes were detected in the A549 cells after treatment of the Mix .α-SMA positive staining were observed in some of the treated A549 cells. It was shown that HMGB34367RNAi has an ability to specifically block the induction ofα-SMA at protein or gene level.Conclusion1,Induction of HMGB1 plays an important role in the process of lung fibrosis2,The truncated form of HMGB1, HMGB34367, can act as a transcriptional factor for up-regulation of expression ofα-SMA.gene, contributing to epithelial cell to myofibroblast transdifferentiation.3. HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF.