Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis

Abstract

BackgroundSpermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’) identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1.MethodsThe testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis.ResultsThe ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter.ConclusionsThe results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma

“…HIF-1α has been reported to play important roles in regulation of MCL-1 expression [32, 33]; although HIF-1α mediated regulation of MCL-1 has not been studied in esophageal adenocarcinoma. ChIP assay was performed using Pierce™ agarose ChIP kit (Thermo Scientific, Rockford, IL).…”

Apoptotic Cell Death Under Hypoxia

“…Antiapoptotic changes include increase in Bcl-2 (83) and Mcl-1 levels (58,71,89), Bcl-xL induction (16,64,83) (through HIF-1␣ binding to a hypoxia-response element in the Bcl-xL promoter), and decreases in pro-apoptotic Bid (22), Bax (22,83), and Bak levels (83).…”

Effects of lipopolysaccharide-induced inflammation on hypoxia and inflammatory gene expression pathways of the rat testis

“…HIF-1α is expressed by Leydig cells in the normoxic adult rat testis and is unregulated by hypoxia [ 30 ]. We have also demonstrated that myeloid leukemia cell differentiation 1 ( Mcl-1 ) is a target gene for testicular HIF-1 with potentially important roles in antiapoptotic protection of Leydig cells [ 31 ]. Understanding mechanisms of HIF-1 activation under normoxic, hypoxic, and other physiological conditions, such as inflammation, in the testis is of significant interest to investigators interested in the roles of HIF-1 on Leydig cell physiology.…”