Regulation of human luteinizing hormone receptor in ... - Springer Link

Reproductive Medicine and Biology 2008; 7: 11–16

doi: 10.1111/j.1447-0578.2007.00196.x

Review Article Blackwell Publishing Asia

Regulation of hLHR in the ovary

Regulation of human luteinizing hormone receptor in the ovary TAKASHI MINEGISHI,* KAZUTO NAKAMURA, SOICHI YAMASHITA, SADATOMO IKEDA and KAYOKO KOGURE Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, Maebashi, Japan

The luteinizing hormone receptor (LHR) is essential for elevated levels of progesterone to maintain pregnancy during the first trimester; the maintenance of the expression of LHR is a key factor controlling the duration of luteal function. Therefore, as the expression of LHR is most likely to be regulated by the stability of the receptor mRNA at the luteal phase of the human menstrual cycle, we focused on studies examining the stability of mRNA rather than the production of mRNA. In addition, LHR (exon 9), one of the splice variants of human LHR (hLHR), was cloned in the corpus luteum of a patient with a regular menstrual cycle. The results of Western blots using Percoll gradient fractionation indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the

INTRODUCTION

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OLLICULOGENESIS, OVULATION AND subsequent luteinization in the ovary are mediated by endocrine factors of the pituitary–gonadal axis. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are heterodimeric glycoproteins comprised of a common α-subunit and a unique β-subunit. Follicle-stimulating hormone and LH are synthesized in gonadotroph cells of the anterior pituitary and act on their cognate receptors in the gonads. Follicle-stimulating hormone receptors (FSH-Rs) are transmembrane proteins present in the female granulosa cells of growing follicles. Luteinizing hormone receptor (LHR) expression is limited to the surrounding theca cells in pre-antral follicles, and as

*Correspondence: Dr Takashi Minegishi, Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Email: [email protected] Received 20 August 2007; accepted 06 November 2007.

transducing organelle. These results showed that hLHR (exon 9) caused a reduction in the expression of functional receptor number and affected the signaling condition of wild-type hLHR. As the luteal phase progressed hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) was expressed more than hLHR in the late luteal phase. This work reveals the essential function of the regulatory and structural elements involved in human LH receptor splicing, and that hLHR (exon 9) can negatively control the function of wild-type receptors. Moreover, this finding presented a novel mechanism of regulation of LHR in the human corpus luteum. (Reprod Med Biol 2008; 7: 11–16) Key words: human luteinizing hormone receptor, E2, ovary, splicing of human LHR.

development proceeds, the granulosa cells of preovulatory follicles and corpora lutea also express LHRs. In maturing follicles, FSH mediates continued mitotic activity of granulosa cells, and decreased FSH responsiveness is associated with follicular atresia. There is a complex interplay between developing follicles and the pituitary. Follicle-stimulating hormone elicits granulosa cell peptide and steroid hormone production by inducing the expression of inhibin/activin subunits and steroidogenic enzymes. Inhibins and activins are dimeric peptide hormones of the transforming growth factor-β (TGF-β) superfamily and are named for their functions in attenuating and enhancing pituitary FSH production, respectively. Ovulation and luteinization of dominant follicles in response to LH depend on LHR expression upregulated by FSH.

INDUCTION OF LUTENIZING HORMONE RECEPTOR

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OLLICLE-STIMULATING HORMONE REGULATES ovarian steroid production by inducing the expression

© 2008 The Authors Journal compilation © 2008 Japan Society for Reproductive Medicine

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of steroidgenic enzymes.1,2 The expression of P450 aromatase and P450 side chain cleavage (P450scc) enzymes is contingent on FSH signaling in developing follicles, whereas luteinization initiates their constitutive expression.3,4 Ovaries of FSH β–/– mice exhibit a striking downregulation of P450 aromatase and P450scc mRNAs. Findings of normal serum estrogen in FSH β-knockout females and estrous periodicity in FSH receptor (FSHR) knockouts may indicate that a nonovarian compensatory mechanism can function to maintain steroid hormone production. It remains to be established whether the local concentration of steroids is altered in the ovaries of FSH β–/– mice, an intriguing question because of the intrafollicular functions of steroids. Moreover, insulin-like growth factor I (IGF-1) knockout females have attenuated expression of FSHR and demonstrate a pre-antral folliclogenesis block, which is likely to be caused by inadequate FSH signaling. Granulosa cells within arrested follicles in FSH β–/– mice do not express detectable LHR and as a result they may lack the potential for LH-mediated steps toward terminal differentiation. It has been recognized that estradiol increases the actions of FSH for differentiation of granulose cell function,5 including amomatase expression,6 estradiol synthesis, FSHR expression and LHR expression. Mvk, a cytosolic enzyme in the cholesterol biosynthetic pathway, was cloned from rat liver in 1990.7 As the ovary is one of the major steroidogenic endocrine tissues, it is not surprising that Mvk exists and is involved in sterol synthesis in the ovary. In fact, Mvk enzyme activity has already been demonstrated in the ovary.8 Moreover, Menon’s group reported that Mvk was a LHR mRNA binding protein and that Mvk binding to LHR mRNA accelerated the LHR mRNA instability. In the ovary, ovarian steroid hormone production uses both plasma-derived cholesterol and de novo synthesized cholesterol as a precursor.9 Ginter et al. reported that a dominant follicle contains more estradiol than other small follicles, indicating that the synergistic action of FSH and estradiol in a dominant follicle may enhance the induction of LHR more than secondary follicles.10 In the dominant follicle, we speculate that estradiol suppresses Mvk expression to assist the induction of LHR in coordination with other factors, for example, activin, TGF-B and IGF-1, prior to the LH surge. The basal transcriptional activity of the hLHR gene has been examined by Dufau and colleagues in JAR cells and simian virus 40 transformed placental cells.11–13 Similar to the rat LH receptor (rLHR), the promoter region of the hLHR appears to be within the first


176 bp of the 5′-flanking region.12 This region of the hLHR gene contains consensus sequences for two Sp1 sites, three AP-2 sites and one estrogen receptor response element half-site (EREhs). Similar to the rLHR gene, the Sp1 sites of the hLHR gene are also involved in regulating the basal transcription of the gene, and EMSAs suggest binding of both Sp1 and Sp3 to each of the two sites.11 Reporter gene constructs in which one or both sites were disrupted show that they each contribute to basal hLHR gene transcription.11 Upstream of these two Sp1 sites is the EREhs at nt-171. Using a yeast one-hybrid screen, Zhang and Dufau13 identified three nuclear orphan receptors (EAR2, EAR3/COUP-TF1 and TR4) that bind to this EREhs. The EMSAs were used to show that endogenous EAR2 and EAR3/COUP-TF1 from JAR cells and from human testis and TR4 from human testis bind to the hLHR EREhs.13 Functional analyses suggest that the binding of EAR2 and EAR3/COUP-TF1 to the hLHR EREhs repress, whereas the binding of TR4 to this site stimulates, hLHR gene transcription.13,14 These studies suggest that the relative abundance of these three nuclear orphan receptors may be significant in determining the basal transcription of the hLHR gene in different cell types. During the growth and differentiation of granulosa cells in the developing follicles there is an estrogendependent and FSH-dependent acquisition of LHR. These actions of FSH can be mimicked, at least in part, by agents that increase intracellular levels of cAMP. The increase in LHR binding activity during this process is accompanied by an increase in LHR mRNA.15,16 Using granulosa cells cultured from estrogen-primed immature rats, Shi and Segaloff16 demonstrated using nuclear runon assays that FSH or cAMP treatment of these cells causes an approximately 10-fold increase in transcription of the endogenous LHR gene. Although these observations do not rule out a potential role for LHR induction also being mediated by increased LHR mRNA stability, they demonstrate that a cAMP-mediated induction of the LHR gene is clearly important in this process. The increased transcription of the LHR gene in response to FSH or cAMP is not observed until 24 or more hours after treatment, and increases in LHR mRNA and human chorionic gonadotropin (hCG) binding activity display a similar lag time.16 Because primary cultures of rat granulosa sells transfected with a reporter gene construct containing 2.1 kb of 5′-flanking sequence of the rLHR gene respond to cAMP treatment, this system is amenable for use in identifying cis- and trans-acting elements mediating cAMP-dependent induction of the rLHR gene. Thus, cells transfected show incremental



increases in the cAMP-mediated fold induction of reporter gene activity as the length of the 5′-flanking region is extended from –40 bp to –2056 bp, suggesting the presence of multiple cAMP-responsive cis elements.17 Similarly, EMSAs using probes corresponding to overlapping portions of the 2.1-kb 5′-flanking sequence and extracts from control versus cAMP-treated granulosa cells show the presence of multiple complexes whose intensities are increased with cAMP treatment.17 Some of the elements in the rLHR gene mediating cAMP responsiveness have been identified. These include the three Sp1 sites mentioned above (Sp1a at nt –83, Sp1b at nt –103, and Sp1c at nt –174). These three sites have also been shown to bind both Sp1 and Sp3 and to be involved in the basal transcription of the rLHR gene in primary cultures of rat granulosa cells.17 In summary, although considerable progress has been made in our understanding of the mechanisms underlying both basal and hormonal regulation of the transcription of the LHR gene in recent years, much more needs to be done to piece together the role of the many elements and factors that govern the cell-specific expression and hormonal regulation of the LHR mRNA.

Regulation of hLHR in the ovary 13

Figure 1 Expression of human luteinizing hormone receptor (hLHR) and hLHR (exon 9) mRNA in the human corpus luteum. Total RNA samples were prepared from the ovary. Reverse transcription-polymerase chain reaction was carried out for hLHR (801 bp) and hLHR (exon 9) (615 bp) using primers at exon 1 and exon 11 (lane 1, 10-day follicle; lane 2, 14-day follicle; lane 3, 21-day corpus luteum; lane 4, 27-day corpus luteum). The LH receptor gene is coded in chromosome number 2 and its size is approximately 80 kb. Exons 1 to 10 code the N-terminal extracellular portion of the receptor, and the rest of the N-terminal portion, the transmembrane domain and the C-terminal domain are encoded by exon 11.

MAINTENANCE OF THE LH RECEPTOR

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HE PRE-OVULATORY LH surge causes first activation and thereafter desensitization of the LHR in luteinized cells. A model for such desensitization involving adenosine diphosphate ribosylation factor 6 (ARF6) and arrestin 2 was recently proposed by Hunzicker-Dunn et al.3 In this model, the switch of the LHR from an actively signaling module to one uncoupled from stimulatory G protein is considered to be intimately associated with the formation of larger protein aggregates containing self-associated LHRs.18 A marked downregulation of cell surface LHR and its cognate mRNA follows desensitization.15,19,20 The downregulation of LHR mRNA that occurs under these conditions does not result from decreased transcription of the LHR gene, but rather from increased degradation of LHR mRNA (Fig. 1).21 Luteinizing hormone receptor (exon 9), one of the splice variants among hLHR, was cloned in the corpus luteum of a patient with a regular menstrual cycle,22 although the functional meaning of LHR (exon 9) was yet unknown. We detected three transcripts (5.4, 3.6, 2.4 kb) of hLHR mRNA using Northern blot of the human ovary,23 indicating that one may encode LHR (exon 9). We showed that the receptor complexes with hLHR and hLHR (exon 9) reduced the expression level of hLHR in 293 cells, resulting in the attenuation of the

expression of hLHR at the plasma membrane. The results of Western blots using Percoll gradient fractionation indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the transducing organelle. The mutant rhodopsin, G protein-coupled photoreceptor, retained in the endoplasmic reticulum, trapped wild-type rhodopsin, demonstrating that the mutant misfolded rhodopsin molecules might interfere with the maturation of wild-type rhodopsin in the endoplasmic reticulum, allowing it to be eventually degraded.24 A number of truncated receptor variants have recently been described, resulting in the reduction of the full-length receptor expressions by coexpression of truncated and wild receptors.2,25,26 In the case of hLHR, we assume that the receptor complex in the endoplasmic reticulum prevents wild-type hLHR from an association with molecules such as chaperone, previously demonstrated to be involved in the maturation of gonadotropin receptors.27 A recent report lends further credence to this hypothesis25 by showing that a naturally occurring truncated mutant of human chemokine receptor 5 exerts a dominant negative effect on wild-type chemokine receptor 5.



We then examined whether the formation of receptor complexes of hLHR affected the binding affinity for hCG and signaling condition. In the 293 cells expressing hLHR (exon 9), no detectable binding of [125I]hCG was found, indicating that hLHR (exon 9) is not expressed on the cell surface. The coexpression of hLHR (exon 9) with hLHR reduced the number of receptors that expressed hLHR alone, without changing the affinity for [125I]hCG. These results lead us to consider the negative control of receptor function thorough the formation of receptor complexes, including receptor number and signal transduction. Thus, the ability to transduce a hCG signal was measured by quantitating cAMP accumulation in cells incubated with increasing concentrations of hCG. The basal levels of cAMP are quite similar to cells expressing both hLHR and hLHR (exon 9). In contrast, effective concentration 50 (EC50) for a hCG-induced cAMP response is approximately sevenfold higher in cells expressing hLHR and hLHR (exon 9) than in cells expressing hLHR alone, and the maximal response in cells expressing both receptors was significantly reduced when compared with cells expressing hLHR alone. These results showed that hLHR (exon 9) caused a reduction in the expression of functional receptor number and affected the signaling condition of wild-type hLHR. Whaley et al.28 carefully explored the effect of varying levels of β2-adrenergic receptor on the activation of adenylyl cyclase, suggesting that the receptor expression levels could inversely affect the functional properties of the receptor (i.e. an increase in EC50 and a decrease in the maximal response). Based on the data that hLHR (exon 9) stays inside the endoplasmic reticulum, we suspect that the decrease in hCG responsiveness in cells expressing both hLHR and hLHR (exon 9) results from a reduction in hLHR expression at the plasma membrane. Taken together, we hypothesize that exon 9 of hLHR is involved in both receptor insertion to the plasma membrane and hormone binding, based on the fact that exon 9 codes leucine-rich repeats 8–9 and the N-terminal part to hinge region. We found that the 68-kDa immature receptor of hLHR and the 81 kDa immature receptor of hFSHR forms receptor complexes with hLHR (exon 9). Decreases in hLHR and hFSH expression at the cell surface resulted from a reduction in the immunoreactive protein in the 293 cells, as confirmed by Western blot. A co-immunoprecipitation study with hLHR hFSHR and hLHR (exon 9) revealed that these receptors physically form receptor complexes. We propose that the misfolded hLHR (exon 9) forms a receptor association


with wild-type gonadotropin receptors. This phenomenon cannot occur with thyroid-stimulating hormone receptor, categorized in the same subclass of G protein coupled recptor with the large glycosylated extracellular domain. In an reverse transcription-polymerase chain reaction (RT-PCR) experiment, we detected hLHR (exon 9) mRNA from the late follicular phase to the late luteal phase. Therefore, both hLHR and hLHR (exon 9) are expressed during the entire luteal phase. To quantify the mRNA levels of hFSHR, hLHR and hLHR (exon 9), we carried out an RT-PCR. As the luteal phase progressed, hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) was expressed more than hLHR in the late luteal phase. In contrast, hFSHR mRNA levels were markedly decreased after ovulation, which is consistent with previous results from our laboratory.29 This result suggested that hLHR (exon 9) predominantly existed against hFSHR from the postovulation to the early luteal phase. Given that hFSHR forms a receptor complex with hLHR (exon 9) in the physiological condition, this association could negatively regulate hFSHR expression as hLHR (exon 9) is increasingly expressed.4 The clinical significance of the receptor complexes of gonadotropin receptors is still unclear. It is well known that many substances, including LH, hCG and cytokines, can regulate luteal function. Further study is required to verify whether the receptor complexes between gonadotropin receptors and hLHR (exon 9) contribute to regulation of the functions and various pathological conditions of the ovary (Fig. 2).

CONCLUSION

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HE MECHANISMS BY which these factors regulate the expression of FSHR mRNA and LHR mRNA are complex. Regulation might be dependent on gene transcription and/or receptor mRNA stability. The expression of FSHR is mainly controlled by the gene transcription, probably because the follicles must become responsive to FSH in a competitive manner, and is affected by intraovarian growth factors. In contrast, the LHR is essential for elevated levels of progesterone to maintain pregnancy during the first trimester; the maintenance of the expression of LHR is a key factor controlling the duration of luteal function. Therefore, the expression of LHR might be regulated by the stability of the receptor mRNA. The findings presented herein show that hFSHR and hLHR are associated with hLHR (exon 9) and that the receptor complexes between wild-type receptors and hLHR (exon 9) can negatively



Regulation of hLHR in the ovary 15

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Figure 2 Regulation of the expression of human luteinizing hormone receptor (hLHR). Upper panel shows the changes in the relative amount of hLHR mRNA during the menstrual cycle (columns) and the dots indicate the hLHR (exon 9)/ hLHR ratios at each point. Lower panel shows our hypothesis that LHR expression is mainly translocated to the Golgi where the sugar chain is converted to the mature state, and finally transported to the plasma membrane. Human LHR is dimerized with immature wild-type receptor and would be delivered to the lysome.

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control the function of wild-type receptors. Further study is required to verify whether the receptor complexes between gonadotropin receptors and hLHR (exon 9) contribute to the regulation of the functions and various pathological conditions of the ovary.

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