Interleukin-1α dependent survival of cardiac fibroblasts is associated with StAR/STARD1 expression and improved cardiac remodeling and function after myocardial infarction.

AIMS: One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI. METHODS AND RESULTS: Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts. CONCLUSIONS: This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI.

Systems genetics identifies Hp1bp3 as a novel modulator of cognitive aging.

An individual's genetic makeup plays an important role in determining susceptibility to cognitive aging. Identifying the specific genes that contribute to cognitive aging may aid in early diagnosis of at-risk patients, as well as identify novel therapeutics targets to treat or prevent development of symptoms. Challenges to identifying these specific genes in human studies include complex genetics, difficulty in controlling environmental factors, and limited access to human brain tissue. Here, we identify Hp1bp3 as a novel modulator of cognitive aging using a genetically diverse population of mice and confirm that HP1BP3 protein levels are significantly reduced in the hippocampi of cognitively impaired elderly humans relative to cognitively intact controls. Deletion of functional Hp1bp3 in mice recapitulates memory deficits characteristic of aged impaired mice and humans, further supporting the idea that Hp1bp3 and associated molecular networks are modulators of cognitive aging. Overall, our results suggest Hp1bp3 may serve as a potential target against cognitive aging and demonstrate the utility of genetically diverse animal models for the study of complex human disease.

Proportionate Dwarfism in Mice Lacking Heterochromatin Protein 1 Binding Protein 3 (HP1BP3) Is Associated With Alterations in the Endocrine IGF-1 Pathway.

Heterochromatin protein 1 binding protein 3 (HP1BP3) is a recently described histone H1-related protein with roles in chromatin structure and transcriptional regulation. To explore the potential physiological role of HP1BP3, we have previously described an Hp1bp3(-/-) mouse model with reduced postnatal viability and growth. We now find that these mice are proportionate dwarfs, with reduction in body weight, body length, and organ weight. In addition to their small size, microcomputed tomography analysis showed that Hp1bp3(-/-) mice present a dramatic impairment of their bone development and structure. By 3 weeks of age, mice of both sexes have severely impaired cortical and trabecular bone, and these defects persist into adulthood and beyond. Primary cultures of both osteoblasts and osteoclasts from Hp1bp3(-/-) bone marrow and splenocytes, respectively, showed normal differentiation and function, strongly suggesting that the impaired bone accrual is due to noncell autonomous systemic cues in vivo. One major endocrine pathway regulating both body growth and bone acquisition is the IGF regulatory system, composed of IGF-1, the IGF receptors, and the IGF-binding proteins (IGFBPs). At 3 weeks of age, Hp1bp3(-/-) mice exhibited a 60% reduction in circulating IGF-1 and a 4-fold increase in the levels of IGFBP-1 and IGFBP-2. These alterations were reflected in similar changes in the hepatic transcripts of the Igf1, Igfbp1, and Igfbp2 genes. Collectively, these results suggest that HP1BP3 plays a key role in normal growth and bone development by regulating transcription of endocrine IGF-1 components.

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

HP1BP3 is a novel histone H1 related protein with essential roles in viability and growth.

The dynamic architecture of chromatin is vital for proper cellular function, and is maintained by the concerted action of numerous nuclear proteins, including that of the linker histone H1 variants, the most abundant family of nucleosome-binding proteins. Here we show that the nuclear protein HP1BP3 is widely expressed in most vertebrate tissues and is evolutionarily and structurally related to the H1 family. HP1BP3 contains three globular domains and a highly positively charged C-terminal domain, resembling similar domains in H1. Fluorescence recovery after photobleaching (FRAP) studies indicate that like H1, binding of HP1BP3 to chromatin depends on both its C and N terminal regions and is affected by the cell cycle and post translational modifications. HP1BP3 contains functional motifs not found in H1 histones, including an acidic stretch and a consensus HP1-binding motif. Transcriptional profiling of HeLa cells lacking HP1BP3 showed altered expression of 383 genes, suggesting a role for HP1BP3 in modulation of gene expression. Significantly, Hp1bp3(-/-) mice present a dramatic phenotype with 60% of pups dying within 24 h of birth and the surviving animals exhibiting a lifelong 20% growth retardation. We suggest that HP1BP3 is a ubiquitous histone H1 like nuclear protein with distinct and non-redundant functions necessary for survival and growth.

Ketoconazole inhibits ovulation as a result of arrest of follicular steroidogenesis in the rat ovary.

OBJECTIVE: Ketoconazole (KCZ) is a known inhibitor of steroidogenic P450 enzymes in the adrenal cortex and the gonads. Previous studies examined the potential clinical use of KCZ for attenuation of ovarian response to gonadotropin treatments. This study aimed to use the superovuating rat model to explore the effect of KCZ on ovarian steroidogenesis, follicular function, and development toward ovulation. METHODS: Prepubertal rats were treated with equine chorionic gonadotropin (eCG)/human CG (hCG) resulting in multiple follicular development and ovulation. The effect of KCZ on this model was examined by administration of KCZ-gel formula and subsequent analyses of ovarian steroidogenesis, rate of ovulation, morphometric assessments of follicular parameters, and cell-specific steroidogenic maturation of the treated ovaries. RESULTS: When applied shortly before gonadotropin stimulation, KCZ markedly reduced ovarian progesterone, androstenedione, and estradiol levels down to 18.7, 36.5, and 19.0%, respectively (P < 0.001). A single KCZ-gel administration of 6, 12, and 24 mg/rat resulted in reduction of ovulated ova/ovary down to 8.6 ± 4.9, 5.1 ± 4.3, and 2.4 ± 3.2, respectively, as compared to 13.6 ± 4.4 ova found in the oviduct of control-gel-injected animals (P < 0.001). An alternative protocol made use of small KCZ doses injected in non-gel formula (5 mg/dose/8 hours), commenced with the eCG administration and terminated 24 hours later; this treatment readily inhibited the ovulation rates to 6.6 ± 6.6 as compared to 16.5 ± 4.1 ova/ovary in the control group (P < 0.01). By contrast, KCZ failed to inhibit ovulation if administered 24 hours after eCG injection. Anovulation by KCZ resulted from arrest of follicular development at the stage of 800-840 µm Graafian follicles as compared to 920 µm of peri-ovulatory follicles (OFs) observed in the control group, P = 0.029. In addition, absence of CYP11A1 expression was evident in the granulosa cell layers of the growth-arrested follicles, which also lacked mucified mature cumulus cell complexes. CONCLUSION: These results suggest that KCZ-mediated inhibition of follicular maturation probably results from impaired steroidogenesis at early phase of follicular development toward ovulation. Hence, attenuation of folliculogenesis by KCZ may be harnessed to modulate gonadotropin-ovarian stimulation in fertility treatments.

Selective inhibition of steroidogenic enzymes by ketoconazole in rat ovary cells.

OBJECTIVE: Ketoconazole (KCZ) is an anti-fungal agent extensively used for clinical applications related to its inhibitory effects on adrenal and testicular steroidogenesis. Much less information is available on the effects of KCZ on synthesis of steroid hormones in the ovary. The present study aimed to characterize the in situ effects of KCZ on steroidogenic enzymes in primary rat ovary cells. METHODS: Following the induction of folliculogenesis in gonadotropin treated rats, freshly prepared ovarian cells were incubated in suspension for up to four hours while radiolabeled steroid substrates were added and time dependent generation of their metabolic products was analyzed by thin layer chromatography (TLC). RESULTS: KCZ inhibits the P450 steroidogenic enzymes in a selective and dose dependent manner, including cholesterol side-chain cleavage cytochrome P450 (CYP11A1/P450scc), the 17α-hydroxylase activity of CYP17A1/P450c17, and CYP19A1/P450arom, with IC50 values of 0.3, 1.8, and 0.3 µg/mL (0.56, 3.36, and 0.56 µM), respectively. Unaffected by KCZ, at 10 µg/mL, were the 17,20 lyase activity of CYP17A1, as well as five non-cytochrome steroidogenic enzymes including 3ß-hydroxysteroid dehydrogenase-Δ(5-4) isomerase type 1 (3ßHSD1), 5α-reductase, 20α-hydroxysteroid dehydrogenase (20α-HSD), 3α-hydroxysteroid dehydrogenase (3α-HSD), and 17ß-hydroxysteroid dehydrogenase type 1 (17HSD1). CONCLUSION: These findings map the effects of KCZ on the ovarian pathways of progestin, androgen, and estrogen synthesis. Hence, the drug may have a potential use as an acute and reversible modulator of ovarian steroidogenesis in pathological circumstances.

StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

Infarct-induced steroidogenic acute regulatory protein: a survival role in cardiac fibroblasts.

Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis in the adrenal cortex and the gonadal tissues. This study reveals that StAR is also expressed at high levels in nonsteroidogenic cardiac fibroblasts confined to the left ventricle of mouse heart examined 3 days after permanent ligation of the left anterior descending coronary artery. Unlike StAR, CYP11A1 and 3ß-hydroxysteroid dehydrogenase proteins were not observed in the postinfarction heart, suggesting an apparent lack of de novo cardiac steroidogenesis. Work with primary cultures of rat heart cells revealed that StAR is induced in fibroblasts responding to proapoptotic treatments with hydrogen peroxide or the kinase inhibitor staurosporine (STS). Such induction of StAR in culture was noted before spontaneous differentiation of the fibroblasts to myofibroblasts. STS induction of StAR in the cardiac fibroblasts conferred a marked resistance to apoptotic cell death. Consistent with that finding, down-regulation of StAR by RNA interference proportionally increased the number of STS-treated apoptotic cells. StAR down-regulation also resulted in a marked increase of BAX activation in the mitochondria, an event known to associate with the onset of apoptosis. Last, STS treatment of HeLa cells showed that apoptotic demise characterized by mitochondrial fission, cytochrome c release, and nuclear fragmentation is arrested in individual HeLa cells overexpressing StAR. Collectively, our in vivo and ex vivo evidence suggests that postinfarction expression of nonsteroidogenic StAR in cardiac fibroblasts has novel antiapoptotic activity, allowing myofibroblast precursor cells to survive the traumatized event, probably to differentiate and function in tissue repair at the infarction site.

High mobility group N proteins modulate the fidelity of the cellular transcriptional profile in a tissue- and variant-specific manner.

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1(tm1/tm1), Hmgn3(tm1/tm1), and Hmgn5(tm1/tm1) mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn(tm1/tm1) lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.

Expression and roles of steroidogenic acute regulatory (StAR) protein in 'non-classical', extra-adrenal and extra-gonadal cells and tissues.

The activity of the steroidogenic acute regulatory (StAR) protein is indispensable and rate limiting for high output synthesis of steroid hormones in the adrenal cortex and the gonads, known as the 'classical' steroidogenic organs (StAR is not expressed in the human placenta). In addition, studies of recent years have shown that StAR is also expressed in many tissues that produce steroid hormones for local use, potentially conferring some functional advantage by acting via intracrine, autocrine or paracrine fashion. Others hypothesized that StAR might also function in non-steroidogenic roles in specific tissues. This review highlights the evidence for the presence of StAR in 17 extra-adrenal and extra-gonadal organs, cell types and malignancies. Provided is the physiological context and the rationale for searching for the presence of StAR in such cells. Since in many of the tissues the overall level of StAR is relatively low, we also reviewed the methods used for StAR detection. The gathered information suggests that a comprehensive understanding of StAR activity in 'non-classical' tissues will require the use of experimental approaches that are able to analyze StAR presence at single-cell resolution.

Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3', 5'-monophosphate dependent and independent regulation.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Levels of steroidogenic acute regulatory protein and mitochondrial membrane potential in granulosa cells of older poor-responder women.

OBJECTIVE: To compare mitochondrial function in granulosa cells obtained from older (>40 y) low-responder IVF patients with that of young (<35 y) good-responder patients. DESIGN: Prospective laboratory research. SETTING: In vitro fertilization unit in a university hospital. PATIENT(S): Twenty patients undergoing IVF treatment cycles. INTERVENTION(S): Ultrasound guided oocytes pick-up. MAIN OUTCOME MEASURE(S): Mitochondrial function examined by using JC-1 stain for the mitochondrial membrane potential in granulosa cells of both groups and Western blots for assaying and quantification of steroidogenic acute regulatory protein (StAR) and p450scc (side-chain cleavage). RESULT(S): The number of granulosa cells per follicle differed between the two groups, with fewer granulosa cells isolated in the older low-responder women, compared with in the young, normal responders who were the control women. Trypan blue-negative cells showed similar undisturbed mitochondrial membrane potential, and similar ratios of apoptotic granulosa cells were observed in the two groups. In addition, there was no difference in StAR and P450scc protein levels between the two groups. CONCLUSION(S): Our results demonstrate a significant decrease in the number of total aspirated granulosa cells per follicle in older, poor-responder women, which probably explains the reduced hormonal production by those follicles. However, those cells demonstrate normal mitochondrial membrane potential as well as similar levels of StAR, P450scc, and de novo steroid hormone synthesis in the two groups of patients. Our results do not support mitochondrial dysfunction as a main mechanism of reproductive aging.

Complex role of the mitochondrial targeting signal in the function of steroidogenic acute regulatory protein revealed by bacterial artificial chromosome transgenesis in vivo.

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors.

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

Transcriptional regulation of the cholesterol side chain cleavage cytochrome P450 gene (CYP11A1) revisited: binding of GATA, cyclic adenosine 3',5'-monophosphate response element-binding protein and activating protein (AP)-1 proteins to a distal novel cluster of cis-regulatory elements potentiates AP-2 and steroidogenic factor-1-dependent gene expression in the rodent placenta and ovary.

The first and key enzyme controlling the synthesis of steroid hormones is cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate overlooked modes of regulation of P450scc transcription in the rodent placenta and ovary. Transcription of P450scc requires two clusters of cis-regulatory elements: a proximal element (-40) known to bind either activating protein 2 (AP-2) in the placenta, or steroidogenic factor 1 in the ovary, and a distal region of the promoter (-475/-447) necessary for potentiation of the AP-2/steroidogenic factor 1-dependent activity up to 7-fold. In primary cultures of mouse trophoblast giant cells and rat ovarian granulosa cells, binding of trans-factors to the distal regulatory sequences generated transcriptional activity in a tissue-specific pattern: in the placenta, cAMP response element (CRE)-binding protein 1 (CREB-1) and GATA-2 binding generates promoter activity in a cAMP-independent manner, whereas in ovarian cells, CREB-1 and GATA-4 are required for FSH responsiveness. However, as ovarian follicles advance toward ovulation, elevated Fra-2 expression replaces CREB-1 function by binding the same CRE(1/2) motif. Our findings suggest that upon onset of follicular recruitment, CREB-1 mediates FSH/cAMP signaling, which switches to cAMP-independent expression of P450scc in luteinizing granulosa cells expressing Fra-2. In the placenta, the indispensable role of CREB-1 was demonstrated by use of dominant-negative CREB-1 mutant, but neither cAMP nor Ser133 phosphorylation of CREB-1 is required for P450scc transcription. These observations suggest that placental regulation of P450scc expression is subjected to alternative signaling pathway(s) yet to be found.

Turnover of StAR protein: roles for the proteasome and mitochondrial proteases.

Steroidogenic acute regulatory protein (StAR) is a mitochondrial protein essential for massive synthesis of steroid hormones in the adrenal and the gonads. Our studies suggest that once synthesized on free polyribosomes, StAR preprotein either associates with the outer mitochondrial membrane to mediate transfer of cholesterol substrate required for steroidgenesis, or it is degraded by the proteasome. Proteasome inhibitors can prevent the turnover of StAR preprotein and other matrix-targeted preproteins. Once imported, excessive accumulation of inactive StAR in the matrix is avoided by a rapid turnover. Unexpectedly, mitochondrial StAR turnover can be inhibited by two proteasome inhibitors, i.e., MG132 and clasto-lactacystin beta-lactone, but not epoxomicin. Use of those inhibitors and immuno-electron microscopy data enabled a clear distinction between two pools of intra-mitochondrial StAR, one degraded by matrix protease(s) shortly after import, while the rest of the protein undergoes a slower and inhibitor resistant degradation following translocation onto to the matrix face of the inner membranes.

A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain.

In an attempt to understand what distinguishes severe acute respiratory syndrome (SARS) coronavirus (SCoV) from other members of the coronaviridae, we searched for elements that are unique to its proteins and not present in any other family member. We identified an insertion of two glycine residues, forming the GxxxG motif, in the SCoV spike protein transmembrane domain (TMD), which is not found in any other coronavirus. This surprising finding raises an "oligomerization riddle": the GxxxG motif is a known dimerization signal, while the SCoV spike protein is known to be trimeric. Using an in vivo assay, we found that the SCoV spike protein TMD is oligomeric and that this oligomerization is driven by the GxxxG motif. We also found that the GxxxG motif contributes toward the trimerization of the entire spike protein; in that, mutations in the GxxxG motif decrease trimerization of the full-length protein expressed in mammalian cells. Using molecular modeling, we show that the SCoV spike protein TMD adopts a distinct and unique structure as opposed to all other coronaviruses. In this unique structure, the glycine residues of the GxxxG motif are facing each other, enhancing helix-helix interactions by allowing for the close positioning of the helices. This unique orientation of the glycine residues also stabilizes the trimeric bundle during multi-nanosecond molecular dynamics simulation in a hydrated lipid bilayer. To the best of our knowledge, this is the first demonstration that the GxxxG motif can potentiate other oligomeric forms beside a dimer. Finally, according to recent studies, the stabilization of the trimeric bundle is linked to a higher fusion activity of the spike protein, and the possible influence of the GxxxG motif on this feature is discussed.

Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models.

Steroidogenic acute regulatory protein (StAR) mediates translocation of cholesterol to the inner membranes of steroidogenic mitochondria, where it serves as a substrate for steroid synthesis. Transcription of StAR in the gonads and adrenal cells is upregulated by trophic hormones, involves downstream signaling pathways and a cohort of trans-factors acting as activators or suppressors of StAR transcription. This study suggests that a 21 basepair long sequence positioned at -81/-61 of the murine StAR promoter is sufficient to confer a robust hormonal activation of transcription in ovarian granulosa cells treated with FSH. We show that recombinant GATA-4 and CCAAT/enhancer-binding protein beta (C/EBPbeta) bind to the promoter at -66/-61 and -81/-70 and activate transcription of a reporter gene when co-expressed in heterologous human embryonic kidney 293 (HEK293) cells. In this cell model, C/EBPbeta and GATA-4 synergize in a sequence dependent manner and p300/CBP further maximizes their joint activities. Inhibitors of the transcriptional activators, such as liver-enriched inhibiting protein (C/EBPbeta-LIP), Friend of GATA-4 (FOG-2) protein and the viral E1A protein abolished the respective factor-dependent activities in HEK293 cells. Binding assays suggest that a dual binding of C/EBPbeta and GATA-4 to the promoter depends on the molar ratio of the factors present while demonstrating GATA-4 predominant association with the promoter DNA. This pattern may reflect on StAR expression at the time of corpus luteum formation when C/EBPbeta levels peak, as does StAR expression.

Analysis of trophoblast giant cell steroidogenesis in primary cultures.

Placental progesterone synthesis in humans prevents abortion of the fetus by maintaining uterine quiescence and low myometrial excitability. In rodents, a transient steroidogenic output is observed in the trophoblast giant cells during mid-pregnancy. Although the exact role of this locally produced progesterone is not clear, rodent trophoblast giant cells are an important cell model for studying the regulation of placental steroidogenesis. This chapter describes the methods we developed to analyze the regulation of genes involved in progesterone biosynthesis in miniature cultures of primary trophoblast cells from rodents. These genes include cholesterol side chain cleavage cytochrome P450 (P450scc) and its accessory proteins, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD). To obtain giant cells, uterine implantation sites are sliced in half, and the trophoblast giant cell layers are separated from the surrounding decidua by scraping. Cells can subsequently be separated by gentle enzymatic digestion with trypsin, or collagenase, and plated for further study in vitro. This chapter provides instructions, insights, and comments instrumental for performing in situ visualization of giant cell mRNA and proteins, analyzing enzyme activities, and conducting promoter analyses with a limited number of cells.