![]() These data suggested that Luman expressed in mouse Leydig cells in an age‐dependent increase manner. Luciferase reporter assay results demonstrated that Luman knock‐down upregulated the activity of SF‐1 and Nur‐77 promoters. By contrast, the level of the nuclear receptor SHP decreased. The nuclear receptors SF‐1 and Nur‐77 were significantly increased upon Luman knock‐down in MLTC‐1. Luman knock‐down caused an increase in human chorionic gonadotropin‐stimulated testosterone production in vitro and in vivo. Luman knock‐down significantly upregulated the expression of steroidogenic acute regulatory, cytochrome P450 cholesterol side‐chain cleavage enzymes, 3β‐hydroxysteroid dehydrogenase, and 17‐α‐hydroxylase/C17–20 lyase in MLTC‐1 cells and PLCs. To investigate the physiological function of Luman, experiments were conducted to examine the consequences of short hairpin RNA‐ and small interfering RNA‐mediated Luman knock‐down in MLTC‐1 and PLCs, respectively. The immunofluorescent experiment indicated that Luman was mainly located within the cytoplasm of murine Leydig tumor cells (MLTC‐1) and primary Leydig cells (PLCs). Luman was not detected in Sertoli cells within the seminiferous tubules at any developmental period. Herein, immunohistochemistry results showed that Luman was specifically expressed in mouse Leydig cells in an age‐dependent increase manner, from prepuberty to sexual maturation. ![]() Luman, also known as cAMP‐response element‐binding protein 3, is an endoplasmic reticulum stress‐related protein that has been identified as a novel transcriptional coregulator of a variety of nuclear receptors. These data indicate that LZIP binds to the C/EBP element and induces CCR2 mRNA and protein. Competition analysis showed that the LZIP binding complex competed with the unlabeled LZIP binding probes ( Figure 3B), indi- cating that the LZIP binding activity is specific. To determine the specificity of LZIP binding activity, nuclear extracts were incubated with the labeled LZIP binding probes in the absence and presence of a 20-fold molar excess of unlabeled LZIP binding probes. Cells transfected with LZIP showed an increase in the formation of this com- plex. ![]() Figure 3A shows a shifted LZIP nuclear complex bound to the probe. To investigate the me- chanism of CCR2 mRNA expression, we perfor- med an electrophoretic mobility shift assay (EMSA) using a 32 P-labeled C/EBP element as a probe. LZIP increased mRNA expressions of CCR2 LZIP binds to the C/EBP element in CCR2 promoter CCR2 promoter contains the CCAAT enhancer binding protein (C/EBP) element (Yamato et al., 1999), and LZIP binds to the C/EBP element and activates transcription from C/EBP-containing repor- ter genes ( Lu et al., 1997).
0 Comments
Leave a Reply. |