Studies of Cxcl12 in Hypoxia-Induced Pulmonary Fibrosis

Chronic lung diseases including asthma and idiopathic pulmonary fibrosis (IPF) are characterized by the airway inflammation, airway remodeling, subepithelial fibrosis, and hypoxia. Previous study indicated that hypoxia plays a critical role in tissue fibrosis. In chronic asthma and IPF, the CXCR4/CXCL12 (stromal cell-derived factor-1, SDF-1) axis plays important role in pulmonary fibrosis. CXCL12 is a potent chemokine for homing of CXCR4+ fibrocytes to sites of lung tissue injury, which directly contribute to pulmonary fibrosis. Circulating CXCR4+ fibrocytes and CXC12 were found to be significantly increases in both plasma and lung of the patient with pulmonary fibrosis. Moreover, an anti-CXCL12 neutralizing antibody attenuated bleomycin-induced pulmonary fibrosis in mice. In addition, CXCL12 plays an important role in carcinoma-associated fibroblast differentiation. These results suggest that interfering with CXCL12 network may help to block pulmonary fibrosis. There is increasing evidence indicated that p300 plays critical role in pulmonary fibrosis. Previous study demonstrated that CXCL12 expression is mediated by hypoxia-inducible factor-1and c/EBPupon hypoxia stimulation, however, whether p300 is involved in hypoxia-induced CXCL12 expression in lung fibroblasts still unknown. Recent study has demonstrated that the migration of fibrocytes in response to CXCL12; however, it remains unknown whether CXCL12 plays a role in hypoxia-induced lung fibroblast differentiation and pulmonary fibrosis. In our preliminary data showed that hypoxia induced CXCL12 release, CTGF and -smooth muscle actin (-SMA, a myofibroblast marker) expression in lung fibroblasts. Moreover, CXCL12 can induce CTGF and -SMA expression in lung fibroblasts. Hypoxia-induced CXCL12 release was inhibited by p300 inhibitor, anacardic acid. The Central Hypothesis is that CXCL12 mediates hypoxia-induced lung fibroblast differentiation and pulmonary fibrosis. Specific Aim 1 (1st year): To establish PI3K/Akt dependent p300 activation and c/EBPmediate hypoxia-induced CXCL12 expression in lung fibroblasts Hypothesis 1: The PI3K/Akt dependent p300 and c/EBPcontribute to hypoxia-induced CXCL12 expression 1.1. Using pharmacological inhibitors and siRNA technology to examine the roles of PI3K, Akt, p300, and c/EBPin hypoxia-induced CXCL12 expression 1.2. To assess the Akt, p300, and c/EBPphosphorylation and activity upon hypoxia stimulation 1.3. To study the roles of PI3K, Akt, p300, c/EBP, and CXCL12 on hypoxia-induced fibrocyte and pleural mesothelial cells chemotaxis by transwell assay (in collaboration with sub-projects 1 and 4) 1.4. To analyze the expression of endothelin (ET-1) and CTGF upon hypoxia stimulation in fibrocyte, lung fibroblasts and pleural mesothelial cells (in collaboration with sub-projects 1 and 4) Specific Aim 2 (2nd year): To characterize the CXCL12 is required for hypoxia-induced lung fibroblast differentiation into myofibroblasts Hypothesis 2: The CXCL12 contributes to hypoxia-induced lung fibroblast differentiation 2.1.To identify expression of CTGF and -SMA in lung fibroblasts by CXCL12 and hypoxia stimulation 2.2.To study the PI3K/Akt and other signal pathways in lung fibroblast differentiation by CXCL12 and hypoxia stimulation 2.3.To study the role of miRNA-155 in CXCL12- and hypoxia-induced lung fibroblast differentiation (in collaboration with sub-project 3) Specific Aim 3 (2nd ～ 3rd years): To study the role of CXCL12 in ovalbumin (OVA)- and bleomycin-induced mouse model of lung fibrosis Hypothesis 3: The CXCL12 mediates OVA- and bleomycin-induced lung fibrosis 3.1.To confirm the levels of CXCL12 and fibrocyte in BAL fluid in OVA- and bleomycin-treated mice 3.2.Using pharmacological inhibitors to study the role of PI3K and p300 on CXCL12, CTGF, and -SMA expression and fibrocyte contents in OVA- and bleomycin-induced mouse model of lung fibrosis 3.3.To examine the CTGF, ET-1, miRNA-155, collagen, and -SMA expression in lung tissue in OVA- and bleomycin-treated conditional CXCL12 knockout mice (in collaboration with sub-projects 1 and 3)