Hypoxia-induced aberrant lactate generation and shuttling serves as a direct signal to promote pro-fibrotic fibroblast phenotypes - Project Summary: Relative hypoxia in the expanded alveolar interstitum in fibrotic lung disease can drive altered fibroblast metabolism characterized by decreased mitochondrial respiration and increased lactate generation. Fibrotic lung fibroblasts demonstrate aberrant phenotypes including persistent differentiation, increased matrix production, apoptosis-resistance, and senescence, which are critical to the pathobiology of progressive fibrotic lung disease. However, our understanding of the mechanistic links between hypoxia, lactate, fibroblast phenotypes and lung fibrosis, is limited. Lungs from patients with IPF (a common and severe form of progressive lung fibrosis) and from mice with experimental lung fibrosis have increased lactate levels. Inhibition of lactate generation, either by preventing pyruvate formation from glucose or by blocking the conversion of pyruvate to lactate, diminishes experimental lung fibrosis in murine models. But the mechanisms by which lactate directly contributes to lung fibrosis remain unclear. We have shown that hypoxia differentially affects the lactate production in normal and IPF fibroblasts through downregulation of the “B” isoform of lactate dehydrogenase-B (LDHB, which decreases lactate by catalyzing conversion back to pyruvate) in IPF, but not normal, fibroblasts. Combined with an increase in the LDHA isoform, this suppression of LDHB amplifies lactate generation by IPF cells under hypoxic conditions. Our preliminary data now show that IPF fibroblasts in hypoxic conditions also have an increase in the lactate export protein MCT4, which promotes lactate transfer to the extracellular space. We have shown that lactate signals through its cognate receptor, GPR-81, to induce normal fibroblast differentiation under hypoxic conditions. We now show that this direct effect of lactate is mediated by a reduction in intracellular cAMP. Our in vivo data show that MCT4 and GPR-81 are increased in the lungs of mice following bleomycin-induced lung injury. MCT4 and GPR-81 are strongly expressed in the fibrotic interstitium and fibroblastic foci in IPF lungs, whereas LDHB is identified in normal, but not IPF lung tissue. Finally, we show that GPR-81 inhibition can diminish bleomycin-induced lung fibrosis in mice. In this proposal, we will examine 1) how hypoxia induces changes in LDHB, MCT4 and GPR-81 expression to regulate lactate generation, shuttling, and signaling; 2) how lactate directly drives pro-fibrotic fibroblast phenotypes (differentiation, proliferation, matrix synthesis, survival, mitochondrial function and senescence) via GPR-81 dependent signaling in a hypoxic microenvironment, and 3) how systemic inhibition or mesenchymal- cell specific deletion of MCT4 and GPR-81 (alone and in combination) impacts the resolution of lung injury and fibrosis. These novel studies will enhance our understanding of how hypoxia-mediated aberrant lactate production, shuttling and signaling directly contributes to lung fibrosis while interrogating the potential to target lactate-mediated signaling as a therapeutic approach to fibrotic lung diseases including IPF.
