The steroidogenic acute regulatory (STAR) protein is an absolute requirement for the transfer of cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone, thus initiating the synthesis of steroid hormones. This transfer of cholesterol is considered the rate-limiting step in the synthesis of steroid hormones in steroidogenic cells. The induction of the STAR protein following treatment of cells with steroidogenic stimuli is characterized by extremely rapid phases of both transcription and translation. Following the cloning and characterization of the Star gene structure in 1994 (1), 1 of the first endeavors in the further study of this gene was to determine the factors that were involved in regulating its rapid expression. Because it was known that STAR expression and steroidogenesis used the cyclic adenosine monophosphate (cAMP) signaling pathway in which protein kinase A (PKA) is activated, the earliest studies concentrated on the search for known PKA-activated transcription factors to determine if they target the promoter region and induce transcription of this gene. The 5′–flanking region of the Star gene was shown to contain a number of consensus sequences for the binding of specific protein transcription factors, and thus, several of these factors were studied in greater detail. The use of various length promoter sequences joined to luciferase reporter genes allowed for the rapid determination of the ability of a number of known transcription factors to activate the Star gene as well as their approximate location within the promoter. In general, it was found that full transcriptional potential of the Star gene was contained within the first 151 nucleotides of the Star promoter region. Predictably, 1 of the first transcription factors studied was steroidogenic factor 1 (SF-1). SF-1 is an orphan nuclear receptor that plays a key role in the expression of many cAMP-regulated genes that are involved in steroidogenic functions as well as in adrenal and gonadal development (2). The first such functional SF-1 binding motif was determined to be at position −135 in the Star promoter (3). Following that initial investigation, several other functional SF-1 binding sites were also characterized (4–6) and found to be capable of inducing transcription of Star. There followed a great deal of additional activity on the characterization of the Star promoter and, as a result, in addition to SF-1, many other protein factors were shown to be involved in the regulation of Star expression. The regulation of Star was also found to be positive in the cases of factors such as CCAAT/enhancer binding protein (C/EBP) (7, 8); a zinc finger family transcription factor member (SP1) (5); the sterol regulatory element binding protein (9, 10); a member of the GATA family of transcription factors (GATA4) (11, 12); the cyclic adenosine monophosphate response element binding protein (13, 14); members of the activator protein 1 transcription factor family, (fos and jun) (15, 16), and the liver-X receptor–retinoid X receptor/retinoic acid receptor complex (17). There also emerged transcription factors that were able to negatively regulate Star promoter activity, as shown in studies on dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X-chromosome (gene 1) (18, 19), yin yang factor 1 (20), and the transcription factor forkhead box O3 (21). Together, all of these factors were shown to interact directly or in complexes with other proteins with specific sequences in the Star promoter and either positively or negatively regulate its expression.
In addition to cAMP-mediated regulation of Star gene expression and steroidogenesis, a number of studies have shown that there also exist several other mechanisms that can regulate Star expression. These pathways are not major pathways in the induction of Star and steroid expression, but rather minor pathways that could serve to more finely regulate steroidogenesis and are often demonstrated to produce less than 1% of the steroid response seen with maximal levels of cAMP-induced pathways. Some non-cAMP pathways that affect steroid production include growth factors such as epidermal growth factor (22, 23) and insulin growth factor 1 (24), which are capable of altering expression levels of STAR protein without increasing intracellular cAMP. Other factors such as chloride ion (Cl−) (25) and calcium ion (Ca2+) (26), have also been shown to alter steroid production in steroidogenic Leydig cells but appear to do so by enhancing the activity of other steroidogenic stimuli, such as luteinizing hormone, indicating that they are not interacting directly with the Star promoter. Similar results were found with arachidonic acid (AA) metabolites, in that blocking AA release in Leydig cells through inhibition of phospholipase A2 activity reduced luteinizing hormone–stimulated steroid synthesis. Once again, there is no indication that the AA metabolites acted directly on the Star promoter (27–29). In summary, the production of low levels of steroids can be achieved in the absence of increased cAMP and often occurs by enhancement of the activity of other steroidogenic stimuli. Another unique mechanism in the regulation of STAR protein expression can be found in the rapid induction of 2 key factors involved in this process. It was shown that PKA can rapidly induce the serine/threonine kinase, salt-inducible kinase 1, and the RNA binding protein Znf3611/Tis11b (30). Salt-inducible kinase 1 was demonstrated to reduce the activity of cyclic adenosine monophosphate response element binding protein regulated transcription coactivator 2, which is a mediator of Star transcription and splicing, whereas Znf3611/Tis11b inhibits translation and controls the removal of the 3.5-kb Star messenger RNA (mRNA) by endonuclease activity. Thus, both of these mechanisms result in attenuation of STAR protein expression, one at the level of transcription and the other at the level of translation.
However, the most efficient means of promoting high levels of steroid production in steroidogenic cells appears to be through the activation of the cAMP signaling pathway to activate transcription factors, which often act in concert with other cofactors, to rapidly and maximally increase Star transcription. This results in increased Star translation and cholesterol delivery to the inner mitochondrial membrane for the initiation of steroidogenesis. As a result, to date, essentially all of the factors that maximally stimulate Star expression act on the Star promoter and affect its expression at the level of transcription (Fig. 1). This situation has appeared to change recently with the work of Men et al (31).
Schematic showing core elements involved in the control of STAR expression at the transcriptional level, and the newly discovered regulation by noncoding RNAs at the posttranscriptional level. Genetic locus not drawn to scale, and positions of transcription factor binding sites are approximate. ATP, adenosine triphosphate; CRE1, cyclic adenosine monophosphate response element 1; CRE2, cyclic adenosine monophosphate response element 2; CRE3, cyclic adenosine monophosphate response element 3; CRTC2, cyclic adenosine monophosphate response element binding protein regulated transcription coactivator 2; DAX-1, dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X-chromosome, gene 1; FOXO3, forkhead box O3; GATA, DNA sequence guanine, adenine, thymine, adenine; SREBP, sterol regulatory element binding protein; YY1, yin yang factor 1.
In their study, Men et al (31). have shown that both the human STAR and mouse Star genes can be regulated by the noncoding RNAs H19 and let-7. Their results demonstrate that STAR/Star mRNA is targeted by the microRNA let-7, resulting in the posttranscriptional inhibition of STAR expression. Moreover, the long-noncoding RNA H19, by binding and sequestering let-7, could modulate its availability and regulate STAR protein expression (Fig. 1). These observations represent the first report of STAR expression being regulated by noncoding RNA. H19 antagonism of let-7 was a recent discovery (32) that could explain effects on different let-7 targets connected to a variety of tissues that have been linked to pluripotency, embryonic development, metabolism, and cancer (33–35). The fact that H19 was also found to be present in reproductive tissues (36) allowed the authors to reason that the H19/let-7 axis might be involved in regulating steroidogenesis. On the basis of microRNA target prediction, the authors identified that STAR and Star mRNAs contained consensus let-7 binding sequences in their 3′ untranslated region, which were conserved in human, monkey, mouse, and rat tissues. Utilizing human granulosa cells as a model system, the authors showed that H19 overexpression resulted in a fourfold increase in expression of STAR mRNA and a 2.3-fold increase in STAR protein. It was also demonstrated that cells transfected with let-7 showed a significant decrease in STAR mRNA expression, indicating that let-7 could act directly on the STAR transcript. The experiments in this study were well performed and the data are clear as proof of concept. The transfection experiments have worked well, and the fact that the let-7 sequence is well conserved in other species strengthens the argument that it may be a universal mechanism in the regulation of STAR expression. It is a novel finding in that it represents the first description of noncoding RNA and microRNA involvement in the regulation of STAR expression.
The most compelling question that can be asked concerning this phenomenon is whether the H19/let-7 axis plays an in vivo physiological role in the regulation of STAR expression and hence, steroidogenesis. Given that let-7 has at least 50 other experimentally verified targets and many more predicted targets, it is important to examine additional H19/let-7–mediated effects and their relevance in steroidogenic cells. What factors might serve to regulate the in vivo induction of H19 or let-7, respectively, and are these inductions tied to steroidogenesis? It would be most informative to test several steroidogenic factors such as trophic hormones or cAMP analogs on these cells and measure H19, let-7, and STAR/Star mRNA, and STAR protein levels. Although the machinery to produce these noncoding RNAs are present in steroidogenic cells, it does not necessarily follow that these mechanisms for regulating STAR expression function in vivo. Also, if they do indeed function in vivo, what is the amount of steroid that can be stimulated by the H19/let-7 axis in comparison to what is seen with maximal trophic hormone stimulation? Therefore, although they are novel, considerable work to ascertain the physiological relevance of these findings lies ahead.
Abbreviations:
- AA
arachidonic acid
- cAMP
cyclic adenosine monophosphate
- C/EBP
CCAAT/enhancer binding protein
- mRNA
messenger RNA
- PKA
protein kinase A
- SF-1
steroidogenic factor 1
- STAR
steroidogenic acute regulatory.
Acknowledgments
Disclosure Summary: The authors have nothing to disclose.
References
Author notes
For article see page
Address all correspondence and requests for reprints to: Douglas M. Stocco, PhD, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th St, 5B108 HSC Bldg., Lubbock, Texas 79430. E-mail: [email protected].
