This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sekar, N. Articles by Veldhuis, J. D. Search for Related Content PubMed PubMed Citation Articles by Sekar, N. Articles by Veldhuis, J. D.

Concerted Transcriptional Activation of the Low Density Lipoprotein Receptor Gene by Insulin and Luteinizing Hormone in Cultured Porcine Granulosa-Luteal Cells: Possible Convergence of Protein Kinase A, Phosphatidylinositol 3-Kinase, and Mitogen-Activated Protein Kinase Signaling Pathways

Division of Endocrinology and Metabolism, Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, M.D., Endocrine Division, Box 202, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908. E-mail: jdv{at}virginia.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60–80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 µM). Excess sterol markedly (50–70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol-repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 µM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH’s contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70–85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 µM), or of mitogen-activated protein kinase kinase, PD 98059 (50 µM), U0126 (10 µM), or the latter’s inactive derivative, U0124 (10 µM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60–80%.

In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
STEROID HORMONES in the ovary, testis, and adrenal gland are synthesized from cholesterol substrate. The latter can be derived by hydrolysis of intracellular cholesteryl ester stores (1), de novo synthesis in the endoplasmic reticulum via acetyl coenzyme A, and/or cellular uptake of high density lipoprotein (HDL)- and/or low density lipoprotein (LDL)-associated sterol (2). Species such as the human, pig, monkey, cow, and, to a lesser extent, rat use substantial amounts of blood-borne cholesterol acquired by the LDL receptor pathway (3, 4, 5, 6, 7, 8, 9). In human preovulatory Graafian follicles, LDL receptor expression increases in granulosa-luteal cells and in the corpus luteum, as assessed by immunohistochemistry and in situ molecular hybridization (3). Likewise in in vitro cultures of granulosa and luteal-like cells, FSH and LH stimulate LDL receptor protein and gene expression (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Notably, cotreatment with trophic peptides, such as insulin or insulin-like growth factor I (IGF-I), can augment this action of gonadotropins supraadditively (10, 11, 12). However, the basis for such bihormonal regulation of LDL receptor gene expression is not known.

Cholesterol and other oxysterols control LDL receptor gene transcription through a negative feedback mechanism (13). Thus, excessive amounts of free sterol repress, whereas cholesterol depletion up-regulates, this gene (11). However, whether gonadotropins and insulin/IGF-I supraadditively relieve sterol autorepression has not been defined. LDL receptor gene transcription is also modulated by sterol-independent effectors, such as growth factors (11, 12, 14, 15, 16), activators of PKA (11, 12) or protein kinase C (PKC), inhibitors of protein synthesis (17, 18, 19), and several cytokines (20, 21, 22).

As one physiologically relevant model in which to investigate the multihormonal regulation of LDL receptor gene expression, we have used an in vitro primary monolayer culture system of porcine granulosa-luteal cells, wherein the gonadotropin, LH, and the growth factor-like peptides, and insulin/IGF-I act supraadditively (12, 23). Because both classes of hormones play critical roles in promoting steroidogenic cytodifferentiation (7, 24, 25, 26), the mechanisms subserving their convergent actions are important to establish. Accordingly, the present experiments explore the hypothesis that LH and insulin/IGF-I drive LDL receptor expression in granulosa-luteal cells synergistically via transcriptional effects with or without preserved sensitivity to sterol feedback. Concurrently, we assessed the mediatory roles of LH-stimulated PKA and insulin/IGF-I-directed phophatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways in bihormonal regulation of this gene.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Ovine LH (NIDDK oLH-26; potency, 2.3 x NIH oLH-S1) and FSH (NIDDK oFSH-19; potency, 94 x NIH oFSH-S1) were obtained from the National Hormone and Pituitary Program, NIH (Bethesda, MD); porcine insulin, human recombinant IGF-I, forskolin, 8-bromo-cAMP (8 Br-cAMP), and 25-hydroxycholesterol were purchased from Sigma (St. Louis, MO); Eagle’s MEM, penicillin/streptomycin, gentamicin, FBS, and Lipofectamine reagent were purchased from Life Technologies, Inc. (Grand Island, NY); wortmannin, LY 294002, PD 98059, U0126, and U0124 were obtained from Calbiochem (La Jolla, CA); and the Dual-Luciferase Reporter Assay System wherein pRL-thymidine kinase (pRL-TK) contains the complementary DNA (cDNA) encoding Renilla reniformis (sea pansy) was obtained from Promega Corp. (Madison, WI).

Cell culture
Porcine ovaries from prepubertal (60–70 kg) gilts were collected from an abattoir and transported in iced saline. The aspirated granulosa cells were cultured to maximize hormonal responsiveness, as previously described (12, 23). Briefly, granulosa cells were isolated from small and medium-sized (1–5 mm) antral follicles by fine needle aspiration under sterile conditions. Cells were washed three times by low speed centrifugation (3000 rpm) in Eagle’s MEM. Approximately 5 x 10⁶ viable granulosa cells were plated in 12-well culture dishes (Corning, Inc., Corning, NY) containing bicarbonate-buffered MEM and 3% FBS (Life Technologies, Inc.) plus insulin (1 µg/ml), estradiol (0.5 µg/ml), and FSH (5 ng/ml) to permit cell anchorage and partial lutein-like maturation. Cells were allowed to attach to culture dishes for 48 h at 37 C and in 5% CO₂.

Transfection of porcine granulosa-luteal cells
We previously cloned a 1076-bp 5'-upstream regulatory fragment (-1076 to +11 bp) of the porcine LDL receptor and generated 5'-nested deletional constructs driving a cytoplasmically targeted firefly luciferase cDNA (11). Transient transfection of reporter constructs was carried out using Lipofectamine (Life Technologies, Inc.). Granulosa cells were plated in 12-well plates and allowed to attach and cytodifferentiate for 48 h (as described above). Medium was changed after the first 24 h. After an additional 24 h, granulosa-luteal cell cultures were rinsed with serum-free MEM without antibiotics for 20–30 min before transfection. Transfection medium (1 ml/well) consisted of serum-free MEM without antibiotics with 2 µg total plasmid DNA (1.8 µg pLDLR/luc and 0.2 µg pRL-TK/luc) and 12 µl Lipofectamine. After transfection for 6 h (based on preliminary optimization experiments), the medium was replaced with serum-free MEM containing antibiotics and the indicated hormone(s), inhibitors, or vehicle. Expression was allowed to proceed for 4 h, which was optimal to define the relative effects of LH and or insulin/IGF-I on the full-length promoter construct (below). Where indicated, granulosa-luteal cells were pretreated for 30 min with 25- hydroxycholesterol or inhibitors of PI 3-kinase or mitogen-activated protein kinase kinase (MEK), and then exposed to LH and or insulin/IGF-I for 4 h in serum-free MEM. Thereafter, cells were rinsed once at room temperature with Dulbecco’s PBS, lysed in 100 µl 1 x lysis buffer (Dual-Luciferase Assay System, Promega Corp.), and stored at -70 C until later assay. Transfection efficiency was monitored by cotransfection of pRL-TK/luc, a vector expressing Renilla luciferase. Data are thus expressed as the ratio of firefly to Renilla luciferase activity. Luciferase activity was measured using 100 µl each of firefly and Renilla luciferin substrate (Promega Corp.) per 20 µl cellular lysate in a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA).

Transfections with 5'-deletional fragments extending from the upstream (-753, -455, -255, and -139 bp) from the transcriptional start site were performed using equimolar concentration of pLDLR/luc constructs. A promoterless luciferase construct, p0/luc, exhibiting no significant activity in response to any intervention, was used to adjust total DNA to 2 µg when necessary. Cotransfection consisted of 1.6 µg full-length LDL receptor promoter (pLDLR1076/luc) with 0.2 µg Rous sarcoma virus (RSV)/protein kinase inhibitor (PKI), 0.2 µg RSV/PKImut, or 0.2 µg p0/luc and pRL-TK/luc. The expression vector for RSV/PKI contained a 251-bp DNA fragment encoding the complete amino acid sequence of rabbit muscle PKI, whereas RSV/PKImut contained oligonucleotides coding for glycine rather than arginine at positions 20 and 21 of the protein (27, 28). A negative control for RSV promoter activity consisted of 0.2 µg RSV/ß-galactosidase, 1.6 µg pLDLR1076/luc, and 0.2 µg pRL-TK/luc.

Statistical methods
Data are presented as the mean ± SEM of three or more independent experiments, each of which was based on a separate batch of 200–300 ovaries to confirm the reproducibility of results. Data were subjected to ANOVA. Means were contrasted by the post-hoc Tukey multiple comparison test. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Initial analyses were carried out to verify the time course of maximal hormonally stimulated transcriptional activity of the transfected full-length (pLDLR1076/luc) LDL receptor promoter and 5'-nested deletional constructs. To this end, transfected granulosa-luteal cells were exposed to vehicle, LH (100 ng/ml), and/or insulin (100 ng/ml) or IGF-I (100 ng/ml) for 4, 24, and 48 h. Incremental (ratio of hormonally stimulated to basal) luciferase activity was maximal at 4 h. Basal activity continued to rise gradually thereafter. Insulin and IGF-I alone stimulated pLDLR1076/luc reporter activity consistently by 1.5- to 2-fold at 24 h, whereas all hormonal effects vanished at 48 h (data not shown). Thus, further studies used the 4-h point to monitor the concerted actions of LH with or without insulin or IGF-I.

Figure 1 summarizes the 8- to 10-fold stimulatory effect of LH alone on pLDLR1076/luc reporter activity observed at 4 h. Whereas insulin and IGF-I alone did not consistently increase luciferase-monitored promoter expression at this time point, combining insulin or IGF-I with LH enhanced pLDLR1076/luc activity further by approximately 2-fold over that achieved by LH per se. To identify possible hormonally responsive DNA regulatory regions, granulosa- luteal cells were transfected with 5'-nested deletional (-753, -455, -255, and -139 bp) LDL receptor constructs. Stimulation by LH alone as well as by LH and insulin/IGF-I was equivalent for full-length pLDL1076/luc and the -753 and -455 bp LDL receptor 5'-deletional promoter constructs (Fig. 1). We could not identify any differential control of the latter three 5'-upstream fragments (-1076, -753, and -455 bp). In contrast, basal and hormonally stimulated LDL receptor transcription depended strongly on the region -255 to -139 bp upstream of the transcriptional start site. Further deletion to the LDL receptor promoter region (-139 to +11 bp) abolished measurable responses.

View larger version (48K): [in this window] [in a new window]   Figure 1. Effects of LH and/or insulin (or IGF-I) on expression of the full-length (-1076 to +11 bp) and selected 5'- deletional fragments of the 5'-upstream putative promoter of the porcine LDL receptor gene driving a cDNA encoding a cytoplasmically localizing luciferase in cultured swine granulosa-luteal cells. After in vitro attachment and lutein-like maturation, cells were Lipofectamine-transfected for 6 h with equimolar amounts of full-length or progressive 5'-deletional LDL receptor promoter fragments, as described in Materials and Methods. Thereafter, granulosa-luteal cells were exposed to vehicle (control), LH (100 ng/ml), and/or insulin or IGF-I (100 ng/ml) for 4 h. Data represent the mean ± SEM of three separate experiments (each performed in duplicate) using TK-driven Renilla (sea pansy) luciferase to normalize transfectional efficiency. Within each transfection set, means with different alphabetic superscripts are significantly different. a, b, and c, P < 0.05.

View larger version (46K): [in this window] [in a new window]   Figure 2. Effects of 8 Br-cAMP (A; 1 mM), forskolin (B; 10 µM), and/or insulin (or IGF-I; 100 ng/ml each) on luciferase expression driven by the full-length (-1076 to +11 bp) and 5'-deletional fragments (-255 or - 139 bp) of the porcine LDL receptor promoter in granulosa-luteal cells. Data represent the mean ± SEM of three separate experiments. Transfection was carried out, followed by agonist exposure as described in Fig. 1. Within each transfection set, means with different alphabetic superscripts are significantly different. a, b, and c, P < 0.05.

View larger version (33K): [in this window] [in a new window]   Figure 3. Effects of exogenous soluble oxysterol on LDL receptor promoter reporter (pLDLR1076/luc) expression. Granulosa-luteal cells were cotransfected pLDLR1076/luc and a Renilla vector, exposed to 25-hydroxycholesterol (25-OH cholesterol; 1 or 10 µM) for 30 min, and then stimulated with control solvent, LH (100 ng/ml), and/or insulin or IGF-I (100 ng/ml) for 4 h. Data represent the mean ± SEM of three separate experiments. Within each transfection set, means with different alphabetic superscripts are significantly different. a, b, and c, P < 0.05.

View larger version (39K): [in this window] [in a new window]   Figure 4. Inhibition of LH-induced LDL receptor promoter (pLDL1076/luc) transcriptional activity by a PKA inhibitor (PKI) expression vector. Granulosa-luteal cells were cultured as described in Materials and Methods. The pLDLR1076/luc reporter vector was cotransfected with an expression plasmid containing either the full coding sequence for the wild-type or mutant PKI driven by the RSV promoter (RSV/PKI or RSV/PKI mut). Cells were subsequently stimulated with control solvent, LH (100 ng/ml) alone, and/or insulin or IGF-I (100 ng/ml) for 4 h. Data represent the mean ± SEM of three separate experiments. Within each transfection set, means with different alphabetic superscripts are significantly different. a, b, and c, P < 0.05.

View larger version (48K): [in this window] [in a new window]   Figure 5. Possible roles of PI 3-kinase and MAPK pathways in the actions of LH and/or insulin/IGF-I. Granulosa- luteal cells were cultured and transfected with pLDLR1076/luc reporter, as described in Materials and Methods. Thereafter, cultures were pretreated for 30 min with two PI 3-kinase-specific inhibitors [A; wortmannin (100 nM) and LY 294002 (10 µM)] or two MEK inhibitors [B; PD 98059 (50 µM) or active U0126 (10 µM), or the latter’s inactive derivative U0124 (10 µM)] before stimulation with LH and/or insulin (or IGF-I) for 4 h. Data represent the mean ± SEM of three separate experiments. Within each transfection set, means with no common superscripts are significantly different. a, b, and c, P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present investigation explores the mechanisms by which LH and insulin/IGF-I act in concert to stimulate LDL receptor gene expression in primary cultures of porcine granulosa-luteal cells. By transient transfection assays, we could identify a putative cis- acting DNA region located -255 to -139 bp 5'-upstream of the transcriptional start site of the homologous LDL receptor gene, which conferred transcriptional responsiveness to LH alone as well as to combined LH-insulin/IGF-I. Concomitant signaling studies indicated that the PKA, PI 3-kinase, and MAP kinase pathways probably participate in LH- and insulin (or IGF-I)-regulated LDL receptor gene expression in this system.

In vivo studies in the pig indicate that LDL receptor gene expression is induced by lutropic hormones and declines upon luteal regression (5). Likewise, in human preovulatory follicles, the LDL receptor transcript was expressed in virtually all granulosa-luteal cells, and apolipoprotein B from LDL particles was detected in granulosa cells located near the basal lamina (3). In in vitro cultures of porcine granulosa-luteal cells, insulin/IGF-I and FSH/LH stimulated LDL receptor messenger RNA accumulation and LDL receptor number and internalization activity (7, 11, 12, 29). CG and 8 Br-cAMP also induced LDL receptor gene expression in human granulosa cells (6, 30), as did FSH (or cAMP analogs) in rat granulosa cells (8). The present documentation of prominent individual and joint control of LDL receptor gene transcriptional activity by LH and insulin/IGF-I extends this concept mechanistically. These findings are especially pertinent, because the abundance of cellular LDL receptors is controlled primarily at the gene transcriptional level (31, 32).

Earlier investigations in nonsteroidogenic cell lines disclosed important transcriptional control of the LDL receptor gene by a 10-bp (5 '-ATCACCCCAC-3 ') sequence located in the so-called second repeat sequence of the proximal 5'-upstream flanking region (33). This motif has been designated sterol regulatory element 1 (SRE-1), because it is activated by conditional positive enhancer proteins, SRE-binding proteins (SREBPs). In particular, withdrawal of free sterol repressors leads to proteolytic release of SREBP’s anchored in the endoplasmic reticulum, their nuclear targeting, and subsequently enhanced LDL receptor gene transcription in liver cells, fibroblasts, and lymphoma cells (34, 35, 36). The present transient transfection analysis in normal granulosa-luteal cells reveals that exposure to 25-hydroxycholesterol likewise can strongly repress basal as well as LH- and LH- plus insulin/IGF-I-stimulated LDL receptor promoter expression. Although SREBPs have not been isolated to date from healthy gonadal steroidogenic cells, homology between the porcine and human LDL receptor would be consistent with the idea of SREBP-dependent autoregulation. In the human LDL receptor gene promoter, two of three imperfect repeats also contain Sp1-binding sites that support basal transcriptional activity and interact with the SRE-1 to amplify gene expression in the low sterol environment (34, 37, 38, 39, 40, 41). Other putative cis-acting DNA elements, such as the novel regulatory element footprint 1 (42), CCAAT/enhancer-binding protein-ß, a functional cAMP-responsive element (22), and yin yang-1 may also confer trans-activational control of this gene (43). In this regard, the present analysis of LDL receptor promoter control in steroidogenically active granulosa-luteal cells localizes the LH and LH/insulin (IGF-I) effect to a region between -255 to -139 bp, which contains both an SRE-1 and two Sp1 sequences (11).

Exposure of granulosa-luteal cells to LH alone for 4 h stimulated porcine LDL receptor gene transcriptional activity by approximately 8- to 10-fold over basal; this inductive effect was mimicked by 8 Br-cAMP and forskolin. These two PKA agonists also achieved a supraadditive interaction with insulin/IGF-I. In one other study 8 Br-cAMP did not stimulate the activity of the human LDL receptor promoter transfected into JEG-3 choriocarcinoma cells (30), whereas (Bu)₂cAMP acted synergistically with oncostatin M in HepG2 cells (22). These apparent distinctions probably reflect different host cellular contexts. In contrast, using primary cultures of untransformed ovarian cells and homologous LDL receptor promoter expression, we show that cotransfection of a PKI minigene, but not a mutant PKI plasmid (RSV/PKImut), significantly attenuates LH-induced pLDLR1076/luc transcriptional activity. As PKI is a heat-stable inhibitor of PKA (27), which facilitates nuclear export of PKA catalytic subunits, thereby limiting transcriptional responses (44, 45), the present data support a pivotal role for the PKA signaling pathway in LH-stimulated LDL receptor gene expression in porcine granulosa-luteal cells.

Under the present culture conditions, acute (4-h) exposure to insulin or IGF-I alone did not stimulate pLDLR1076/luc activity in maturing granulosa-luteal cells, similar to earlier findings using immature granulosa cells (11). Interestingly, long-term (24-h) exposure to insulin/IGF-I increased LDL receptor promoter expression consistently by 1.5- to 2-fold (data not shown). This difference between 4 and 24 h responses could reflect the delayed time dependency of insulin-IGF-I actions recognized in other systems, such as the human LDL receptor promoter expressed in HepG2 cells (16). Even so, at 4 h we observed consistently positive LH-insulin/IGF-I interactions in granulosa-luteal cells, wherein addition of insulin/IGF-I amplified the maximal effect of LH by 2-fold. This interaction was supraadditive, thus defining true synergy between LH and insulin/IGF-I at the transcriptional level.

The insulin-related signaling mechanisms that induce LDL receptor gene transcription in gonadal cells are not understood. In HepG2 (tumoral liver) cells, the p42/44 MAP kinase (extracellular signal-regulated kinase 1/2) cascade up-regulates LDL receptor gene expression, whereas the p38MAP kinase -isoform is repressive (46). Interestingly, in the HepG2 cell line, p42/44 MAP kinase-induced phosphorylation of serine residues in SREBP1a/SREBP2 may have been involved in mediating activation of the LDL receptor promoter by insulin and platelet-derived growth factor, whereas PI 3-kinase was not required (47). On the other hand, in primary cultures of hepatocytes, activation of PKB (a downstream target of PI 3-kinase activation) stimulated SREBP activity, apparently without dependence on p42/44 MAP kinase (48). Thus, existing literature points to important target cell specificity of the signaling and effector role(s) of PI 3-kinase, PKB, and certain MAP kinases in the control of LDL receptor gene expression. In cultured granulosa cells, putative suppression of MAP kinase activity with PD98059 (49) and PI 3-kinase with wortmannin or LY 294002 (50) triggered in vitro apoptosis. Conversely, in luteal cells, insulin and IGF-I increased PI 3-kinase activity and DNA synthesis (51), and in granulosa cells, FSH, forskolin, and 8 Br-cAMP stimulated phosphorylation of PKB by PI 3-kinase-sensitive and PKA-insensitive pathways (52). Accordingly, our inference of an evident dependence of the joint actions of LH and insulin on PI 3-kinase and MAP kinase signaling in granulosa-luteal cells extends the foregoing proposed signaling mechanisms to include transcriptional control activity of the LDL receptor gene. In this regard, other recent studies have shown that IGF-I can activate PI 3-kinase (50), and FSH/LH can stimulate MAP kinase (53) in pig granulosa cells.

In relation to LDL receptor gene regulation in other tissues overexpression of upstream activators of the MAPKs, such as MEKK1 or MEK1, can induce LDL receptor promoter activity by severalfold in an SRE-1-related manner (47). Conversely, overexpression of MKK6(E), a constitutive activator of p38 MAPK, significantly repressed LDL receptor promoter expression (46). Thus, certain stimuli may up-regulate this gene by way of p42/44 MAP kinase activation (54) and/or phosphorylation of SREBP-1 (55). As indicated above, both the type of hormonal agonist and the cell context seem to influence the relative roles of the PI 3-kinase and MAP kinase pathways in modulating SREBP’s production and/or activation (48). The present studies are noteworthy in pointing to both PI 3-kinase and p42/44 MAP kinase involvement in LDL receptor gene control by LH/insulin (IGF-I) in granulosa-luteal cells, but do not exclude or identify possible roles for PKB or p38 MAP kinases. As we show that concurrent activation of the PKA pathway is required for synergistic (bihormonal) induction of porcine LDL receptor gene expression in granulosa-luteal cells, further studies in the present model of transcriptionally responsive ovarian cells should generate new insights into convergent control of this promoter by PKA, PI 3-kinase, and MAP kinase signaling pathways.

	Acknowledgments

 
We thank Drs. Holly A. LaVoie for earlier preparation of the LDL receptor promoter fragments, and Richard N. Day (University of Virginia, Charlottesville, VA) for RSV/PKI and RSV/PKImut expression vectors.

Received February 21, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Veldhuis JD, Strauss 3rd JF, Silavin SL, Kolp LA 1985 The role of cholesterol esterification in ovarian steroidogenesis: studies in cultured swine granulosa cells using a novel inhibitor of acyl coenzyme A: cholesterol acyltransferase. Endocrinology 116:25–30[Abstract/Free Full Text]
2. Miller WL 1988 Molecular biology of steroid hormone synthesis. Endocr Rev 9:295–318[Abstract/Free Full Text]
3. Yamada S, Fujiwara H, Kataoka N, Honda T, Nakayama T, Higuchi T, Mori T, Maeda M 1998 Stage-specific uptake of apolipoprotein-B in ovarian follicles and corpora lutea of the menstrual cycle and early pregnancy. Hum Reprod 13:944–952[Abstract/Free Full Text]
4. Grummer RR, Carroll DJ 1988 A review of lipoprotein cholesterol metabolism: importance to ovarian function. J Anim Sci 66:3160–3173
5. LaVoie HA, Benoit AM, Garmey JC, Dailey RA, Wright DJ, Veldhuis JD 1997 Coordinate developmental expression of genes regulating sterol economy and cholesterol side-chain cleavage in the porcine ovary. Biol Reprod 57:402–407[Abstract]
6. Chaffin CL, Dissen GA, Stouffer RL 2000 Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys. Mol Hum Reprod 6:11–18[Abstract/Free Full Text]
7. Veldhuis JD, Nestler JE, Strauss JF, 3rd, Gwynne JT 1986 Insulin regulates low density lipoprotein metabolism by swine granulosa cells. Endocrinology 118:2242–2253[Abstract/Free Full Text]
8. Reaven E, Tsai L, Spicher M, Shilo L, Philip M, Cooper AD, Azhar S 1994 Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions. J Cell Physiol 161:449–462[CrossRef][Medline]
9. Soumano K, Price CA 1997 Ovarian follicular steroidogenic acute regulatory protein, low-density lipoprotein receptor, and cytochrome P450 side-chain cleavage messenger ribonucleic acids in cattle undergoing superovulation. Biol Reprod 56:516–522[Abstract]
10. Garmey JC, Day RN, Day KH, Veldhuis JD 1993 Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: in vitro studies with swine granulosa cells. Endocrinology 133:800–808[Abstract/Free Full Text]
11. LaVoie HA, Garmey JC, Day RN, Veldhuis JD 1999 Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: promoter activation, messenger ribonucleic acid stability, and sterol feedback. Endocrinology 140:178–186[Abstract/Free Full Text]
12. Sekar N, Garmey JC, Veldhuis JD 2000 Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells: joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (stAR) protein and cytochrome P450 side-chain cleavage (P450scc) enzyme. Mol Cell Endocrinol 159:25–35[CrossRef][Medline]
13. Hussain MM, Strickland DK, Bakillah A 1999 The mammalian low-density lipoprotein receptor family. Annu Rev Nutr 19:141–172[CrossRef][Medline]
14. Nicholson AC, Hajjar DP 1992 Transforming growth factor-beta up-regulates low density lipoprotein receptor-mediated cholesterol metabolism in vascular smooth muscle cells. J Biol Chem 267:25982–25987[Abstract/Free Full Text]
15. Parini P, Angelin B, Lobie PE, Norstedt G, Rudling M 1995 Growth hormone specifically stimulates the expression of low density lipoprotein receptors in human hepatoma cells. Endocrinology 139:3767–3773
16. Streicher R, Kotzka J, Muller-Wieland D, Siemeister G, Munck M, Avci H, Krone W 1996 SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I. J Biol Chem 271:7128–7133[Abstract/Free Full Text]
17. Auwerx JH, Chait A, Wolfbauer G, Deeb SS 1989 Involvement of second messengers in regulation of the low-density lipoprotein receptor gene. Mol Cell Biol 9:2298–2302[Abstract/Free Full Text]
18. Kumar A, Chambers TC, Cloud-Heflin BA, Mehta KD 1997 Phorbol ester-induced low density lipoprotein receptor gene expression in HepG2 cells involves protein kinase C-mediated p42/44 MAP kinase activation. J Lipid Res 38:2240–2248[Abstract]
19. Makar RS, Lipsky PE, Cuthbert JA 2000 Multiple mechanisms, independent of sterol regulatory element binding proteins, regulate low density lipoprotein gene transcription. J Lipid Res 41:762–774[Abstract/Free Full Text]
20. Liu J, Shoyab M, Grove RI 1993 Induction of Egr-1 by oncostatin M precedes up-regulation of low density lipoprotein receptors in HepG2 cells. Cell Growth Differ 4:611–616[Abstract]
21. Li C, Kraemer FB, Ahlborn TE, Liu J 1999 Induction of low density lipoprotein receptor (LDLR) transcription by oncostatin M is mediated by the extracellular signal-regulated kinase signaling pathway and the repeat 3 element of the LDLR promoter. J Biol Chem 274:6747–6753[Abstract/Free Full Text]
22. Liu J, Ahlborn TE, Briggs MR, Kraemer FB 2000 Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter. J Biol Chem 275:5214–5221[Abstract/Free Full Text]
23. Sekar N, LaVoie HA, Veldhuis JD 2000 Concerted Regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells. Endocrinology 141:3983–3992[Abstract/Free Full Text]
24. Giudice LC 1992 Insulin-like growth factors and ovarian follicular development. Endocr Rev 13:641–669[Abstract/Free Full Text]
25. Adashi EY 1998 The IGF family and folliculogenesis. J Reprod Immunol 39:13–19[CrossRef][Medline]
26. Poretsky L, Cataldo NA, Rosenwaks Z, Giudice LC 1999 The insulin-related ovarian regulatory system in health and disease. Endocr Rev 20:535–582[Abstract/Free Full Text]
27. Day RN, Walder JA, Maurer RA 1989 A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription. J Biol Chem 264:431–436[Abstract/Free Full Text]
28. Maurer RA 1989 Both isoforms of the cAMP-dependent protein kinase catalytic subunit can activate transcription of the prolactin gene. J Biol Chem 264:6870–6873[Abstract/Free Full Text]
29. Veldhuis JD 1988 Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells. Endocrinology 123:1660–1667[Abstract/Free Full Text]
30. Takagi K, Strauss 3rd JF 1989 Control of low density lipoprotein receptor gene expression in steroidogenic cells. Can J Physiol Pharmacol 67:968–973[Medline]
31. Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340[CrossRef][Medline]
32. Brown MS, Goldstein JL 1986 A receptor-mediated pathway for cholesterol homeostasis. Science 232:34–47[Free Full Text]
33. Smith JR, Osborne TF, Goldstein JL, Brown MS 1990 Identification of nucleotides responsible for enhancer activity of sterol regulatory element in low density lipoprotein receptor gene. J Biol Chem 265:2306–2310[Abstract/Free Full Text]
34. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS 1993 SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75:187–197[CrossRef][Medline]
35. Sakai J, Nohturfft A, Goldstein JL, Brown MS 1998 Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies. J Biol Chem 273:5785–5793[Abstract/Free Full Text]
36. Osborne TF 2000 Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action. J Biol Chem 275:32379–32382[Free Full Text]
37. Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS 1993 Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem 268:14497–14504[Abstract/Free Full Text]
38. Wang X, Sato R, Brown MS, Hua X, Goldstein JL 1994 SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77:53–62[CrossRef][Medline]
39. Sanchez HB, Yieh L, Osborne TF 1995 Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. J Biol Chem 270:1161–1169[Abstract/Free Full Text]
40. Yieh L, Sanchez HB, Osborne TF 1995 Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter. Proc Natl Acad Sci USA 92:6102–6106[Abstract/Free Full Text]
41. Bennett MK, Osborne TF 2000 Nutrient regulation of gene expression by the sterol regulatory element binding proteins: increased recruitment of gene-specific coregulatory factors and selective hyperacetylation of histone H3 in vivo. Proc Natl Acad Sci USA 97:6340–6344[Abstract/Free Full Text]
42. Mehta KD, Chang R, Underwood J, Wise J, Kumar A 1996 Identification of a novel cis-acting element participating in maximal induction of the human low density lipoprotein receptor gene transcription in response to low cellular cholesterol levels. J Biol Chem 271:33616–33622[Abstract/Free Full Text]
43. Bennett MK, Ngo TT, Athanikar JN, Rosenfeld JM, Osborne TF 1999 Co-stimulation of promoter for low density lipoprotein receptor gene by sterol regulatory element-binding protein and Sp1 is specifically disrupted by the yinyang 1 protein. J Biol Chem 274:13025–13032[Abstract/Free Full Text]
44. Wiley JC, Wailes LA, Idzerda RL, McKnight GS 1999 Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A. J Biol Chem 274:6381–6387[Abstract/Free Full Text]
45. Zheng XL, Matsubara S, Diao C, Hollenberg MD, Wong NC 2000 Activation of apolipoprotein AI gene expression by protein kinase A and kinase C through transcription factor, Sp1. J Biol Chem 275:31747–31754[Abstract/Free Full Text]
46. Singh RP, Dhawan P, Golden C, Kapoor GS, Mehta KD 1999 One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade. J Biol Chem 274:19593–19600[Abstract/Free Full Text]
47. Kotzka J, Muller-Wieland D, Roth G, Kremer L, Munck M, Schurmann S, Knebel B, Krone W 2000 Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade. J Lipid Res 41:99–108[Abstract/Free Full Text]
48. Fleischmann M, Iynedjian PB 2000 Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt. Biochem J 349:13–17[CrossRef][Medline]
49. Oliver RH, Khan SM, Leung BS, Yeh J 1999 Induction of apoptosis in luteinized granulosa cells by the MAP kinase kinase (MEK) inhibitor PD98059. Biochem Biophys Res Commun 263:143–148[CrossRef][Medline]
50. Westfall SD, Hendry IR, Obholz KL, Rueda BR, Davis JS 2000 Putative role of the phosphatidylinositol 3-kinase-Akt signaling pathway in the survival of granulosa cells. Endocrine 12:315–321[CrossRef][Medline]
51. Chakravorty A, Joslyn MI, Davis JS 1993 Characterization of insulin and insulin-like growth factor-I actions in the bovine luteal cell: regulation of receptor tyrosine kinase activity, phosphatidylinositol-3-kinase, and deoxyribonucleic acid synthesis. Endocrinology 133:1331–1340[Abstract/Free Full Text]
52. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS 2000 Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14:1283–12300[Abstract/Free Full Text]
53. Cameron MR, Foster JS, Bukovsky A, Wimalasena J 1996 Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5'-monophosphates in porcine granulosa cells. Biol Reprod 55:111–119[Abstract]
54. Dhawan P, Bell A, Kumar A, Golden C, Mehta KD 1999 Critical role of p42/44(MAPK) activation in anisomycin and hepatocyte growth factor- induced LDL receptor expression: activation of Raf-1/Mek-1/p42/44(MAPK) cascade alone is sufficient to induce LDL receptor expression. J Lipid Res 40:1911–1919[Abstract/Free Full Text]
55. Roth G, Kotzka J, Kremer L, Lehr S, Lohaus C, Meyer HE, Krone W, Muller-Wieland D 2000 MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro. J Biol Chem 275:33302–33307[Abstract/Free Full Text]

This article has been cited by other articles:

				L. R. Soria, S. A. Gradilone, M. C. Larocca, and R. A. Marinelli Glucagon induces the gene expression of aquaporin-8 but not that of aquaporin-9 water channels in the rat hepatocyte Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1274 - R1281. [Abstract] [Full Text] [PDF]

				J.-H. Choi, C.-L. Chen, S. L. Poon, H.-S. Wang, and P. C K Leung Gonadotropin-stimulated epidermal growth factor receptor expression in human ovarian surface epithelial cells: involvement of cyclic AMP-dependent exchange protein activated by cAMP pathway Endocr. Relat. Cancer, March 1, 2009; 16(1): 179 - 188. [Abstract] [Full Text] [PDF]

				S. Natesampillai, J. Kerkvliet, P. C. K. Leung, and J. D. Veldhuis Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E385 - E391. [Abstract] [Full Text] [PDF]

				J.-H. Choi, A. S. T. Wong, H.-F. Huang, and P. C. K. Leung Gonadotropins and Ovarian Cancer Endocr. Rev., June 1, 2007; 28(4): 440 - 461. [Abstract] [Full Text] [PDF]

				P. C.K. Leung and J.-H. Choi Endocrine signaling in ovarian surface epithelium and cancer Hum. Reprod. Update, March 1, 2007; 13(2): 143 - 162. [Abstract] [Full Text] [PDF]

				C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]

				J M Silva, M Hamel, M Sahmi, and C A Price Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. Reproduction, December 1, 2006; 132(6): 909 - 917. [Abstract] [Full Text] [PDF]

				S. Ledoux, D. B. Campos, F. L. Lopes, M. Dobias-Goff, M.-F. Palin, and B. D. Murphy Adiponectin Induces Periovulatory Changes in Ovarian Follicular Cells Endocrinology, November 1, 2006; 147(11): 5178 - 5186. [Abstract] [Full Text] [PDF]

				S. Natesampillai, M. E. Fernandez-Zapico, R. Urrutia, and J. D. Veldhuis A Novel Functional Interaction between the Sp1-like Protein KLF13 and SREBP-Sp1 Activation Complex Underlies Regulation of Low Density Lipoprotein Receptor Promoter Function J. Biol. Chem., February 10, 2006; 281(6): 3040 - 3047. [Abstract] [Full Text] [PDF]

				J. A. MacLean II, M. K. Rao, K. M.H. Doyle, J. S. Richards, and M. F. Wilkinson Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP Biol Reprod, December 1, 2005; 73(6): 1126 - 1134. [Abstract] [Full Text] [PDF]

				S. Rice, N. Christoforidis, C. Gadd, D. Nikolaou, L. Seyani, A. Donaldson, R. Margara, K. Hardy, and S. Franks Impaired insulin-dependent glucose metabolism in granulosa-lutein cells from anovulatory women with polycystic ovaries Hum. Reprod., February 1, 2005; 20(2): 373 - 381. [Abstract] [Full Text] [PDF]

				R. C. Seals, R. J. Urban, N. Sekar, and J. D. Veldhuis Up-Regulation of Basal Transcriptional Activity of the Cytochrome P450 Cholesterol Side-Chain Cleavage (CYP11A) Gene by Isoform-Specific Calcium-Calmodulin-Dependent Protein Kinase in Primary Cultures of Ovarian Granulosa Cells Endocrinology, December 1, 2004; 145(12): 5616 - 5622. [Abstract] [Full Text] [PDF]

				N. Sekar and J. D. Veldhuis Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells Am J Physiol Endocrinol Metab, July 1, 2004; 287(1): E128 - E135. [Abstract] [Full Text] [PDF]

				G. Zhang and J. D. Veldhuis Requirement for Proximal Putative Sp1 and AP-2 cis-Deoxyribonucleic Acid Elements in Mediating Basal and Luteinizing Hormone- and Insulin-Dependent in Vitro Transcriptional Activation of the CYP17 Gene in Porcine Theca Cells Endocrinology, June 1, 2004; 145(6): 2760 - 2766. [Abstract] [Full Text] [PDF]

				G. Zhang and J. D. Veldhuis Insulin Drives Transcriptional Activity of the CYP17 Gene in Primary Culturesof Swine Theca Cells Biol Reprod, June 1, 2004; 70(6): 1600 - 1605. [Abstract] [Full Text] [PDF]

				D. K. Singh, V. Mokashi, C. L. Elmore, and T. D. Porter Phosphorylation of Supernatant Protein Factor Enhances Its Ability to Stimulate Microsomal Squalene Monooxygenase J. Biol. Chem., February 14, 2003; 278(8): 5646 - 5651. [Abstract] [Full Text] [PDF]

				C. R. O. Carvalho, J. B. C. Carvalheira, M. H. M. Lima, S. F. Zimmerman, L. C. Caperuto, A. Amanso, A. L. Gasparetti, V. Meneghetti, L. F. Zimmerman, L. A. Velloso, et al. Novel Signal Transduction Pathway for Luteinizing Hormone and Its Interaction with Insulin: Activation of Janus Kinase/Signal Transducer and Activator of Transcription and Phosphoinositol 3-Kinase/Akt Pathways Endocrinology, February 1, 2003; 144(2): 638 - 647. [Abstract] [Full Text] [PDF]

				S. L. Lay, I. Lefrere, C. Trautwein, I. Dugail, and S. Krief Insulin and Sterol-regulatory Element-binding Protein-1c (SREBP-1C) Regulation of Gene Expression in 3T3-L1 Adipocytes. IDENTIFICATION OF CCAAT/ENHANCER-BINDING PROTEIN beta AS AN SREBP-1C TARGET J. Biol. Chem., September 13, 2002; 277(38): 35625 - 35634. [Abstract] [Full Text] [PDF]

				Y. Wang, S. Chan, and B. K. Tsang Involvement of Inhibitory Nuclear Factor-{kappa}B (NF{kappa}B)-Independent NF{kappa}B Activation in the Gonadotropic Regulation of X-Linked Inhibitor of Apoptosis Expression during Ovarian Follicular Development in Vitro Endocrinology, July 1, 2002; 143(7): 2732 - 2740. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sekar, N. Articles by Veldhuis, J. D. Search for Related Content PubMed PubMed Citation Articles by Sekar, N. Articles by Veldhuis, J. D.