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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

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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

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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

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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

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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.

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