p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

Abstract

Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α‒HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1αsuppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

Deciphering the Pharmacological Mechanisms of Taohe-Chengqi Decoction Extract Against Renal Fibrosis Through Integrating Network Pharmacology and Experimental Validation In Vitro and In Vivo

“…Fibrosis causes microvasculature injury and inflammatory stimulation decreases oxygen permeation and increases renal tubular epithelial cells metabolism, which can lead to the persistent hypoxic condition of renal tissue. Conversely, hypoxia can trigger the apoptosis of renal tubular epithelial cells and stimulate the phenotype transformation from renal tubular cells to myofibroblasts (Hu et al, 2017;Liu et al, 2018;Shu et al, 2019). Hypoxia could also promote the expression of tissue inhibitors of metalloproteinases (TIMPS), which would inhibit the degradation of ECM, reduce the blood flow of capillaries around renal tubules and damage the diffusion of oxygen, thus aggravating the local hypoxia (Volker, 2009).…”

TGF‐β1–p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications

“…p-p53 S15 is involved in p53 stabilization and transactivation of p53-responsive promoters (including PAI-1), whereas p-p53 S9 participates in p53-SMAD signaling (77,78). Pharmacologic inhibition (with pifithrin-a) or genetic deletion in the proximal tubular epithelium confirmed involvement of p53 in prolonged G 2 /M residence and the fibrotic response to cisplatin, UUO, or ischemic injury (18,21,79,80). Similarly, cisplatin-or bilateral ischemiainduced AKI in streptozotocin-treated mice or genetically susceptible (Akita) diabetic animals is significantly diminished by pifithrin-a, p53 siRNA, or proximal tubuletargeted p53 ablation (81).…”

“…One such coactivator is p53, a sequence-specific DNA-binding protein and a key regulator of the cell growth, apoptosis, and senescence programs as well as the cellular stress response (62)(63)(64)(65). p53 is activated in the injured renal epithelium, initiating cell cycle arrest at the G 1 and G 2 /M checkpoints depending on the participating effectors (e.g., ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related serine/ threonine-protein kinase, casein kinase 1 and 2, p21, and TGF-b1) and the extent of tissue hypoxia (18,63,66). TGF-b1 signaling in the damaged kidney increases p53 levels and phosphorylation, particularly at p53 S9/15 ( Fig.…”

“…Functional impairment following AKI may be transient (adaptive repair), reflecting compensatory regeneration of surviving tubular elements. Severe or persistent injury, however, triggers inflammatory cell infiltration, reduced microvascular density, epithelial dedifferentiation, and G 2 /M growth arrest (11,(16)(17)(18)(19)(20)(21) (Fig. 1A).…”

Expression significance and potential mechanism of hypoxia‐inducible factor 1 alpha in patients with myelodysplastic syndromes

“…Hypoxia‐inducible factor 1 alpha (HIF‐1α), a subunit that maintains the activity of hypoxia‐inducible factor 1 (HIF‐1), is the key regulatory factor inducing the expression of genes related to cell survival and adaptation in hypoxia conditions . HIF‐1 is considered to be important for regulating the oxygen balance in cells and mediating hypoxic reactions; therefore, it is correlated with the onset and development of tumors.…”