Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

Abstract

Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser(133)Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

“…The reaction mix was separated on bis-tris gel and transferred to polyvinylidene difluoride membranes (Invitrogen). The membranes were incubated with V5 antibody (mouse, 1:1000; Thermo Scientific, MA5-15253), MRAP antibody (mouse, 1 mg/ml; Abcam, Cambridge, MA, USA, ab168120), steroidogenic acute regulatory protein (StAR) antibody (rabbit, 1: 2500; kindly provided by Dr D B Hales, Southern Illinois University School of Medicine) (Hales et al 2000), phosho-StAR (P-StAR) antibody (rabbit, 1: 1000, kindly provided by Dr D M Stocco, Texas Tech University Health Sciences Center) (Manna et al 2006), or b-actin antibody (mouse, 1:10,000; A5441, Sigma-Aldrich) ( Table 1) followed by secondary antibody incubation at room temperature. Signals were detected with enhanced electrochemiluminescence kit (Thermo Scientific).…”

Cross-talk between G Protein-coupled and Epidermal Growth Factor Receptors Regulates Gonadotropin-mediated Steroidogenesis in Leydig Cells

“…EGFR Signaling Regulates Steroidogenesis by Activating StARAs mentioned, StAR is an important rate-limiting protein in gonadotropin-induced steroid production, and some evidence suggests that StAR may be regulated by EGFR signaling in gonadal tissues (12,26). To address the relationship between EGFR signaling and StAR activity in MTLC-1 Leydig cells, the effect of the EGFR inhibitor AG1478 on StAR-independent steroidogenesis was examined.…”

Diallyl sulfide protects against N -nitrosodiethylamineinduced liver tumorigenesis: Role of aldose reductase

“…MDA was one of the main lipid peroxidation products; its elevated levels can reflect the degree of lipid-peroxidation-induced injury in hepatocytes [46] . On the other hand, it has been reported that SOD and CAT constitute a mutually supportive defense against ROS [47] . The decreased activity of SOD in liver of NDEA-treated rats may have been due to the enhanced lipid peroxidation or inactivation of the antioxidative enzymes.…”