Tobacco smoking is a well-known risk factor for both fibrogenesis and fibrotic progression; however, the mechanisms behind these processes remain enigmatic. RTKs (receptor tyrosine kinases) have recently been reported to drive profibrotic phenotypes in fibroblasts during pulmonary fibrosis (PF). Using a phospho-RTK array screen, we identified the RTK AXL as a top upregulated RTK in response to smoke. Both expression and signaling activity of AXL were indeed elevated in lung fibroblasts exposed to tobacco smoke, whereas no significant change to the levels of a canonical AXL ligand, Gas6 (growth arrest–specific 6), was seen upon smoke treatment. Notably, we found that smoke-exposed human lung fibroblasts exhibited highly proliferative and invasive activities and were capable of inducing fibrotic lung lesions in mice. Conversely, genetic suppression of AXL in smoke-exposed fibroblasts cells led to suppression of AXL downstream pathways and aggressive phenotypes. We further demonstrated that AXL interacted with MARCKS (myristoylated alanine-rich C kinase substrate) and cooperated with MARCKS in regulating downstream signaling activity and fibroblast invasiveness. Pharmacological inhibition of AXL with AXL-specific inhibitor R428 showed selectivity for smoke-exposed fibroblasts. In all, our data suggest that AXL is a potential marker for smoke-associated PF and that targeting of the AXL pathway is a potential therapeutic strategy in treating tobacco smoking–related PF.

Cigarette smoke has been shown to trigger aberrant signaling pathways and pathophysiological processes; however, the regulatory mechanisms underlying smoke-induced gene expression remain to be established. Herein, we observed that two smoke-responsive genes, HO-1 and CYP1A1, are robustly induced upon smoke by different mechanisms in human bronchial epithelia. CYP1A1 is mediated by aryl hydrocarbon receptor signaling, while induction of HO-1 is regulated by oxidative stress, and suppressed by N-acetylcysteine treatment. In light of a pivotal role of NRF2 and BACH1 in response to oxidative stress and regulation of HO-1, we examined if smoke-induced HO-1 expression is modulated through the NRF2/BACH1 axis. We demonstrated that smoke causes significant nuclear translocation of NRF2, but only a slight decrease in nuclear BACH1. Knockdown of NRF2 attenuated smoke-induced HO-1 expression while down-regulation of BACH1 had stimulatory effects on both basal and smoke-induced HO-1 with trivial influence on NRF2 nuclear translocation. Chromatin immunoprecipitation assays showed that smoke augments promoter-specific DNA binding of NRF2 but suppresses BACH1 binding to the HO-1 promoter ARE sites, two of which at −1.0 kb and −2.6 kb are newly identified. These results suggest that the regulation of NRF2 activator and BACH1 repressor binding to the ARE sites are critical for smoke-mediated HO-1 induction.

The innate and adaptive immunities have been documented to participate in the pathogenesis of nephrotoxic acute kidney injury (AKI); however, the mechanisms controlling these processes have yet to be established. In our cisplatin-induced AKI mouse model, we show pathological damage to the kidneys, with the classical markers elevated, consistent with the response to cisplatin treatment. Through assessments of the components of the immune system, both locally and globally, we demonstrate that the immune microenvironment of injured kidneys was associated with an increased infiltration of CD4+ T cells and macrophages concomitant with decreased Treg cell populations. Our cell-based assays and animal studies further show that cisplatin exposure downregulated the protein levels of programmed death-ligand 1 (PD-L1), an immune checkpoint protein, in primary renal proximal tubular epithelial cells, and that these inhibitions were dose-dependent. After orthotopic delivery of PD-L1 gene into the kidneys, cisplatin-exposed mice displayed lower levels of both serum urea nitrogen and creatinine upon PD-L1 expression. Our data suggest a renoprotective effect of the immune checkpoint protein, and thereby provide a novel therapeutic strategy for cisplatin-induced AKI.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with limited therapeutic options. Cyclin-dependent kinase 9 (CDK9), a key transcriptional regulator, has been implicated in fibrotic diseases, but no therapies targeting CDK9 have been developed for IPF. This investigation found that CDK9 expression was significantly elevated in IPF lung fibroblasts, correlating with an enhanced fibrogenic transcriptional profile and phenotype. Treatment with our designated CDK9 inhibitor, flavopiridol, exhibited significant anti-fibrotic effects, including suppression of fibrotic marker expression, reduction of fibroblast invasion and proliferation in vitro, and inhibition of fibroblastic lesion expansion in precision-cut lung slices ex vivo. In a bleomycin-induced mouse model of lung fibrosis, systemic administration of flavopiridol improved survival, attenuated body weight loss, and reduced fibrotic lesions and collagen deposition, outperforming the FDA-approved drug nintedanib. Further, we developed an inhalable formulation of flavopiridol and demonstrated its efficacy in mitigating fibrosis through local lung delivery, which minimized systemic drug exposure and potential adverse effects. These findings establish CDK9 as a critical regulator of lung fibrosis and provide compelling evidence for developing CDK9 inhibitors as novel therapeutic agents for IPF.