Elements involved in the regulation of the StAR gene.

The steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane, the regulated step in steroidogenesis. A most interesting facet of this protein is the manner in which its expression is acutely regulated. In this regard, a number of studies have concentrated on the search for consensus cis regulatory elements within its promoter, and, more importantly, on whether these elements are involved in its expression. This short review will summarize some of the findings that have been reported concerning the nature of how the expression of this gene is regulated.

The role of arachidonic acid in steroidogenesis and steroidogenic acute regulatory (StAR) gene and protein expression.

This study was conducted to examine the mechanism for arachidonic acid (AA) regulation of steroidogenic acute regulatory (StAR) protein expression and the relationship between AA and cAMP in hormone-induced steroidogenesis. Dibutyryl cyclic AMP (Bt(2)cAMP)-stimulated MA-10 Leydig cells were treated with AA and/or the phospholipase A(2) inhibitor, dexamethasone. Dexamethasone significantly reduced Bt(2)cAMP-stimulated progesterone production, StAR promoter activity, StAR mRNA, and StAR protein. The inhibitory effects of dexamethasone were reversed by the addition of 150 microm AA to MA-10 cells. In addition, MA-10 cells were treated with the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the 5-lipoxygenase inhibitor, AA861, the epoxygenase inhibitor, miconazole, and the cyclooxygenase inhibitor, indomethacin. Both NDGA and AA861 inhibited progesterone production and StAR protein expression. AA861-inhibited progesterone synthesis and StAR protein were partially reversed by addition of the 5- lipoxygenase metabolite, 5(S)-hydroperoxy-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid. Inhibition of epoxygenase activity inhibited progesterone production significantly, but StAR protein was only slightly reduced. Indomethacin enhanced StAR protein expression and significantly increased progesterone production. Inhibition of AA release or lipoxygenase activities did not affect protein kinase A activity, whereas inhibition of protein kinase A activity using H89 reduced Bt(2)cAMP-induced StAR protein. AA alone did not induce StAR protein expression nor steroid production. These results demonstrate the essential role of AA in steroid biosynthesis and StAR gene transcription and suggest the possible involvement of the lipoxygenase pathway in steroidogenesis. This study further indicates that AA and cAMP transduce signals from trophic hormone receptors to the nucleus through two separate pathways and act to co-regulate steroid production and StAR gene expression and indicates that both pathways are required for trophic hormone-stimulated steroidogenesis.

Transcriptional regulation of the StAR gene.

The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step of steroidogenesis. In steroidogenic tissues, the StAR gene is regulated acutely by trophic hormone through a cAMP second messenger pathway. Thus, the gene encoding StAR must be finely regulated so that it is expressed in steroidogenic tissues at the proper time in development, and must be rapidly induced in response to cAMP stimulation. We have summarized the available information concerning the regulation of StAR mRNA levels including promoter mapping and transactivation studies. We also discuss the various transcription factors which have been implicated in the regulation of the StAR gene thus far, and propose models of how StAR transcription may be regulated.

SF-1 (steroidogenic factor-1) and C/EBP beta (CCAAT/enhancer binding protein-beta) cooperate to regulate the murine StAR (steroidogenic acute regulatory) promoter.

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. We have examined the StAR promoter for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Through sequence analysis, deoxyribonuclease I (DNAse I) footprinting and electrophoretic mobility shift assays (EMSAs), we have identified two putative CCAAT/enhancer binding protein (C/EBP) DNA elements at -113 (C1) and -87 (C2) in the mouse StAR promoter. Characterization of these sites by EMSA indicated that C/EBP beta bound with high affinity to C1 and C2 was a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter showed that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-stimulated/basal] of the promoter. Furthermore, we have demonstrated that these two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP beta is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis.

Pregnenolone synthesis in immature rat Sertoli cells.

It has been reported that testicular Sertoli cells can be induced to synthesize the steroidogenic acute regulatory (StAR) protein. StAR mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. Since Sertoli cells are thought to be unable to utilize cholesterol for the synthesis of steroids the role of StAR in these cells was questioned. In the present studies we have corroborated the induction of StAR protein in immature cultured Sertoli cells in response to either trophic hormone or cAMP analog stimulation. Further, we have shown that long term stimulation of Sertoli cells with cAMP analog results in the induction of P450scc enzyme and increased pregnenolone production. In this manner, the Sertoli cell may resemble its ovarian homolog, the granulosa cell, more closely than previously thought with regards to its steroidogenic capacity. Thus, StAR may play the same role in Sertoli cells as it does in other steroidogenic tissues.

POU domain genes are differentially expressed in the early stages after lineage commitment of the PNS-derived stem cell line, RT4-AC.

RT4 is a family of cell lines derived from a rat peripheral neurotumor and consists of a multipotential stem cell which spontaneously gives rise to a glial derivative and two neuronal derivatives. To begin to understand the role(s) of transcription factors in neural differentiation, we examined the expression of ten transcription factor genes (MASH1, REST/NRSF, Oct-1, Oct-2, Tst-1/SCIP, Brn-1, Brn-2, Brn-3.0, Brn-4, Brn-5) in the RT4 cell lines. We report here that all of the RT4 cells express REST/NRSF, Oct-1 and Brn-5, but do not express MASH1, Brn-3.0 or Brn-4. Furthermore, Brn-2 and Tst-1/SCIP expression was restricted to the RT4 stem cell line and glial derivative, while Oct-2 was expressed predominantly by the RT4 stem cell line and neuronal derivatives. We propose that the lack of expression of MASH1 (which is expressed relatively early in autonomic neuron differentiation) and Brn-3.0 (which is expressed early in sensory neuron differentiation), in combination with the presence of REST/NRSF (a repressor of neuronal gene expression), in all of the RT4 cell lines, establishes the RT4 system as a unique model for examining very early events in neuronal versus glial cell fate determination.

Characterization of developmental stage and neuronal potential of the rat PNS-derived stem cell line, RT4-AC.

RT4 is a family of cell lines derived from a rat peripheral neurotumor and consists of a multipotential stem cell line that spontaneously gives rise to three derivative cell types: one glial-like and two neuronal-like. Previous studies have established that the RT4 glial derivative expresses many properties of Schwann cells; however, the neuronal designation of the other RT4 derivatives is less well substantiated. To further characterize the developmental stage and lineages represented by the RT4 stem cell and its derivatives we examined the expression of 16 marker genes whose expression is either specific to neurons or in some cases, neural tissue. Taken together our results indicate that (i) the RT4 neuronal-like derivatives express only immature neuronal properties, (ii) the RT4 cell lines most closely resemble neural crest derivatives from embryonic day 10 to 12 in the rat, (iii) treatment with cAMP and steroids, although capable of promoting process extension by the RT4 neuronal-like derivatives, did not affect the expression of any of the 16 marker genes examined, and (iv) when compared to other neural stem cell systems, RT4-AC generates the most immature neuronal derivatives.

Regulation of the macrophage population in postnatal rat testis.

Testicular macrophages increase in concentration during postnatal development in rats. This process may be under hormonal control since administration of hCG stimulates a similar increase to occur precociously. The purpose of the present studies was to determine how the macrophage population is regulated during normal postnatal development and in response to exogenous hCG. We first determined that testicular macrophages proliferate in situ during development and that hCG administration results in an increase in proliferation when given to 10-day-old rats. We next evaluated whether hCG might exert its effects through enhanced secretion of testosterone from Leydig cells. We found that testosterone could not induce a precocious increment in the macrophage concentration when it was administered to newborn pups for 10 days. Finally, the normal increase in macrophage concentration that occurs prior to puberty could not be blocked by treatment with the antiandrogen Casodex. The results are consistent with the hypothesis that the macrophage population expands by proliferation, perhaps under gonadotropin control. In addition, neither the precocial expansion that occurs in response to hCG nor the normal expansion that occurs before puberty is mediated by testosterone.

Genetic control of murine hematopoietic stem cell pool sizes and cycling kinetics.

Bone marrow from each of two inbred mouse strains, C57BL/6J and DBA/2J, was highly enriched for stem cells using flow cytometry and was divided into two stem cell subpopulations using the mitochondrial dye rhodamine 123 (Rh-123). The Rh-123lo population was determined to be more primitive than Rh-123hi based on the expression of stem cell markers such as the c-kit protooncogene (stem cell factor receptor) and the Ly-6A/E stem cell antigen (Sca-1) as well as the lack of in vitro colony-forming ability. Compared to DBA/2J mice, marrow from the C57BL/6J strain consistently showed a higher proportion of "very primitive" (Rh-123lo) cells, suggesting that the sizes of functionally distinct stem cell subpopulations are maintained under precise genetic control. Marrow from both strains exposed to the cytotoxic drug 5-fluorouracil showed a dramatic increase in the proportion of Rh-123lo cells within 2 days as repopulation began. Marrow subpopulations returned to pretreatment proportions by the eighth day in DBA/2J mice but not until 14 days in C57BL/6J mice. This intrinsic difference in 5-fluorouracil recovery time was attributed to an increase rate of stem cell cycling in DBA/2J relative to C57BL/6J mice. When stem cell factor was injected into a C57BL/6J<-->DBA/2J allophenic mouse, blood cell chimerism shifted markedly but transiently toward the DBA/2J genotype, suggesting that the DBA/2J target population, because of an inherent kinetic advantage, was able to respond faster to the cytokine. A model is proposed that is based on these and our earlier observations to explain this strain-specific stem cell behavior and offer new insights into the genetic control of stem cell cycling and population dynamics.