LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site.

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to -715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by downregulating Cyp19A1 expression.

The transition from granulosa cell (GC) to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 (Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone (LH)/human chorionic gonadotropin treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore, we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory GCs obtained from pregnant mare serum gonadotropin-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG-Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and chromatin immunoprecipitation assays. The results revealed that the steroidogenic factor-1 (SF1)-binding motif, but not the specificity protein 1 (Sp1) binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs and that it has an essential role in the luteal transition in steroidogenesis.

Role of Klf4 in the Regulation of Apoptosis and Cell Cycle in Rat Granulosa Cells during the Periovulatory Period.

In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague⁻Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition.

Effect of radioactive iodine-induced hypothyroidism on longitudinal bone growth during puberty in immature female rats.

Thyroid cancer in children, the most common endocrine malignancy, shows aggressive behavior and has a high recurrence rate after surgical ablation. Radioactive iodine (RAI) treatment is the most effective primary modality for medical ablation of juvenile thyroid cancer, and leads to intentional hypothyroidism. Although several negative impacts of hypothyroidism have been reported in children in response to other antithyroid agents, the combined effects of RAI exposure and hypothyroidism, on growing bones specifically, are unknown. In this study, we investigated the effect of RAI-induced hypothyroidism on the long bones during the pubertal growth spurt using immature female rats. Female Sprague-Dawley rats were randomly divided into a control group, and an RAI-treated group fed with RAI (0.37 MBq/g body weight) twice via gavage. After 4 weeks, we observed a significantly-reduced serum free thyroxine level in the RAI group. The latter group also displayed decreased body weight gain compared to the control. In addition, the lengths of long bones, such as the leg bones and vertebral column, as well as bone mineral content, were reduced in the RAI-treated animals. Our results confirm the negative impacts of RAI-induced thyroid deficiency during puberty on longitudinal bone growth and bone mineralization.

Peri-pubertal high caffeine exposure increases ovarian estradiol production in immature rats.

Chronic caffeine consumption exerts a negligible effect on the reproductive organs of normal adult females, but it is not known whether this is also true for children and adolescents. Here, we investigated the effects of high caffeine exposure on sexual maturation and ovarian estradiol production in immature female rats. Immature female SD rats were divided into controls and caffeine groups fed 120 and 180mg/kg/day for 4 or 8 weeks. There was a significant delay in vaginal opening in the caffeine-fed groups. In addition, serum estradiol levels were elevated in the caffeine-fed animals after 2 and 4 weeks of exposure. Estradiol secretion as well as aromatase expression also increased significantly in the ovarian cells in response to caffeine. These results demonstrate that peripubertal exposure to high caffeine increases estradiol production in the ovary; this may disturb the coordinated regulation of the hypothalamo-pituitary-ovarian axis, thereby interfering with sexual maturation.

Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate.

We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg-1  day-1 for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine-fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1-1 mm, followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage-specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.

Coxsackievirus B3 infection reduces female mouse fertility.

Previously we demonstrated coxsackievirus B3 (CVB3) infection during early gestation as a cause of pregnancy loss. Here, we investigated the impacts of CVB3 infection on female mouse fertility. Coxsackievirus-adenovirus receptor (CAR) expression and CVB3 replication in the ovary were evaluated by immunohistochemistry or reverse transcription-polymerase chain reaction (RT-PCR). CAR was highly expressed in granulosa cells (GCs) and CVB3 replicated in the ovary. Histological analysis showed a significant increase in the number of atretic follicles in the ovaries of CVB3-infected mice (CVBM). Estrous cycle evaluation demonstrated that a higher number of CVBM were in proestrus compared to mock mice (CVBM vs. mock; 61.5%, 28.5%, respectively). Estradiol concentration in GC culture supernatant and serum were measured by an enzyme-linked immunosorbent assay. Baseline and stimulated levels of estradiol in GC were decreased in CVBM, consistent with significantly reduced serum levels in these animals. In addition, aromatase transcript levels in GCs from CVBM were also decreased by 40% relative to the mock. Bone mineral density evaluated by micro-computed tomography was significantly decreased in the CVBM. Moreover, the fertility rate was also significantly decreased for the CVBM compared to the mock (CVBM vs. mock; 20%, 94.7%, respectively). This study suggests that CVB3 infection could interfere with reproduction by disturbing ovarian function and cyclic changes of the uterus.

Wilms' tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells.

BACKGROUND: The important role of WT1 in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of inhibin-α and FSH receptor. There is also considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage. METHODS: GCs were obtained from small preantral follicles of immature rat ovary. Bax and bcl-2 mRNA and protein levels in GCs transfected with WT1 (-KTS) or WT1 (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with caspase 3/7 activity and TUNEL assay. The mechanism by which WT1 regulates Bax expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs. RESULTS: Here, we showed that WT1 (-KTS) suppressed endogenous Bax transcript and protein expression, and this inhibition resulted from direct binding of WT1 in the Bax promoter region in vivo. In addition, anti-apoptotic effects of WT1 (-KTS) were demonstrated based on MTT assays, a sensitive bioluminescence caspase 3/7 assay and TUNEL assays. On the other hand, WT1 has no role on bcl-2 expression in GCs. CONCLUSION: These findings suggest that activation of WT1 is necessary for maintenance of GC survival during early stage of follicles and WT1 can play a role in protecting apoptosis through the regulation of upstream activator (Bax), as well as through regulation of downstream effecter (caspases 3 and 7).

Cervical cytopathological findings in Korean women with Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum infections.

This is to investigate the cervical cytological abnormalities associated with Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum infections on routine screen. A total of 714 subjects who had undergone cervical Pap smears and concomitant analyses for cervical infections were included by a retrospective search. The frequencies of reactive cellular change (RCC) and squamous epithelial abnormalities were significantly higher in Chlamydia positive subjects than in uninfected subjects (P<0.001). Of the 124 subjects tested for M. hominis, M. genitalium, and U. urealyticum, 14 (11%) were positive for M. hominis and 29 (23%) were positive for U. urealyticum. Squamous abnormalities were more frequent in subjects with Ureaplasma infections than in uninfected subjects (24% versus 8%). Taking together these findings, C. trachomatis and U. urealyticum may have a causal role in the development of cervical epithelial changes, including RCC. Thus, extra awareness is warranted in cervical screening of women with Chlamydia or Ureaplasma infections.

Regulation of follicle growth and apoptosis in cultured mouse ovaries by NGF and oxygen.

Cryopreservation of ovarian tissue has been proposed for use in preserving female fertility before anticancer chemo-radiotherapy, because ovarian tissue contains a large pool of non-growing, primordial follicles. The mechanisms that regulate the exit of follicles from the pool are poorly understood. To determine optimal conditions for in vitro ovarian culture, we investigated the effects of nerve growth factor (NGF) and oxygen concentration on follicle growth and apoptosis. Oxygen concentration affected both cell proliferation and apoptosis. Under 20% oxygen, but not 1.5% or 5%, NGF decreased apoptosis in mouse ovaries by down-regulating the pro-apoptotic genes Bax and p53. In conclusion, high oxygen tension during in vitro ovarian culture promotes follicle growth and, in conjunction with NGF, suppresses apoptosis. The efficiency of this method to preserve fertility depends in part on the level of atresia. These results suggest that oxygen and NGF may be used to increase numbers of preantral follicles and mature oocytes in the culture of mammalian ovarian cortical strips.

Downregulation of KLF4 and the Bcl-2/Bax ratio in advanced epithelial ovarian cancer.

Kruppel-like factor 4 (KLF4) is a key transcriptional regulator of cell differentiation and proliferation and an altered expression of KLF4 has been reported in a number of human malignancies. In the present study, we investigated KLF4 expression and its role in cell proliferation in advanced epithelial ovarian cancer (EOC). We compared KLF4, Bcl-2 and Bax transcript levels in ovaries isolated from advanced EOC and normal control ovaries. In addition, the KLF4 gene was transduced into ovarian cancer cells and transcript levels of Bcl-2 and Bax and cell proliferation were analyzed by real-time RT-PCR and MTT assays, respectively. Ovarian KLF4 expression and Bcl-2/Bax ratios were downregulated in most cases of advanced EOC. In addition, KLF4 overexpression in ovarian cancer cells increased the Bcl-2/Bax ratio. However, MTT analysis indicated that the overexpression of KLF4 had no effect on the proliferation of ovarian cancer cells. The inactivation of KLF4 is frequently observed in ovarian cancers and a reduced expression of KLF4 in the ovarian cancers may lead to a reduction in the Bcl-2/Bax ratio. The latter has a role in predicting cancer grade, although its exact role in ovarian carcinogenesis requires clarification.

Ovarian cyst torsion coexisting with acute pyelonephritis in an eight-year-old premenarchal girl.

We report the case of a premenarchal eight-year-old girl with an adnexal torsion accompanied by acute pyelonephritis with vague, non-specific symptoms. In young girls, symptoms may be vague, with patients complaining of non-specific gastro-intestinal problems. Combination with other complications such as pyelonephritis makes the diagnosis of adnexal torsion even more difficult as in the present case. To preserve future reproductive function, early surgical intervention is required. Although the impact on menarchal development of oophorectomy or partial removal of ovarian tissue before menarche remains to be evaluated, timely recognition and treatment of adnexal torsion clearly increases the possibility of retaining the ovary. Therefore, to decrease the time between the onset of symptoms and the identification of patients at risk, sonography should be considered as a first imaging modality for evaluating children with any degree of abdominal pain accompanied by non-specific symptoms.

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

Clinicopathological characteristics of ovarian tumours according to menarchal status in Korean girls.

AIM: To evaluate our experience with ovarian tumours in young girls and to identify specific characteristics in relation to their menarchal status that might assist in the early diagnosis and prompt management. METHODS: Girls younger than 18 years of age who had an adnexal tumour confirmed at surgery from 1985 to 2008 were identified from the pathology database of Hanyang University Medical Centre. Clinicopathological characteristics such as patient's age, age at menarche, presenting symptoms, operative findings and final histological diagnosis were analysed. RESULTS: Twenty-four of the 90 patients operated on for an ovarian tumour were pre-menarchal and 66 were post-menarchal. Most tumours were of germ cell origin: 83% of tumours in pre-menarchal and 53% in post-menarchal girls. Most of the patients complained of abdominal pain (∼70%), although a palpable mass was identified in only 17% of pre-menarchal and 30% of post-menarchal patients. Vomiting was a major complaint in pre-menarchal patients (30%) but was uncommon in post-menarchal patients (<5%). The frequency of torsion was significantly higher in pre-menarchal (50%) than in post-menarchal (17%) patients, and more than 90% of the torsion in pre-menarchal patients occurred in tumours of germ cell origin. CONCLUSIONS: The incidence of ovarian tumour and torsion is rare, especially in pre-menarchal girls. However, we found a higher prevalence of torsion in pre-menarchal girls with an ovarian tumour. A high level of suspicion for torsion should be considered when treating pre-menarchal girls with an ovarian tumour, irrespective of the tumour size to preserve adnexal tissue.

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

Regulation of Wilms' tumor gene expression by nerve growth factor and follicle-stimulating hormone in the immature mouse ovary.

This study investigated the regulation of Wilms' tumor gene (WT1) in the ovary by nerve growth factor and FSH to better understand signals that initiate early follicular growth. Nerve growth factor showed a direct stimulatory effect on endogenous expression of WT1, whereas FSH attenuated basal and nerve growth factor-stimulated WT1 protein expression, which most likely depended on FSH responsiveness according to the follicle growth stage.

Intermedin functions as a pituitary paracrine factor regulating prolactin release.

Calcitonin, alpha- and beta-calcitonin gene-related peptides, amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/calcitonin gene-related peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that IMD is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that IMD could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for IMD in the pituitary, quantitative and in situ hybridization analyses showed the expression of IMD receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with IMD leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, IMD treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with IMD leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of IMD expression in lactating and ovariectomized rats showed that pituitary IMD transcripts in lactating animals increased to more than 2-fold over nonlactating controls whereas ovariectomy leads to a 90% reduction of IMD expression in the pituitary. Of importance, subsequent treatment with 17beta-estradiol or diethylstilbestrol increased pituitary IMD expression in ovariectomized rats. In addition, analysis of the proximate region of the IMD gene promoter showed that the IMD gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that IMD represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.

Intermedin, a novel calcitonin family peptide that exists in teleosts as well as in mammals: a comparison with other calcitonin/intermedin family peptides in vertebrates.

Endocrine regulation in vertebrates is critical for the adaptation and regulation of homeostasis. The G protein-coupled receptor (GPCR) signaling transduction system represents one of the most ancient forms of cell surface signaling. Recently, comparative sequence analysis has aided in the identification and pairing of a variety of ligand/GPCR signaling systems. Among the ligands of type II GPCRs, the calcitonin family peptides including calcitonin, alpha-calcitonin gene-related peptide (alphaCGRP), betaCGRP, adrenomedullin, and amylin are among the best studied hormones, and the founding member, calcitonin, was originally identified and isolated from teleosts. This unique group of peptides shares a conserved tertiary structure with an N-terminal disulfide-bridged ring. In mammals, these peptides signal through two closely related type II GPCRs and three unique receptor activity-modifying proteins. Recently, based on the analysis of multiple vertebrate genomes, we identified a novel calcitonin/CGRP family peptide named intermedin. Here we show that in humans the five paralogous family genes, calcitonin, CGRP, amylin, adrenomedullin, and intermedin, evolved before the emergence of modern vertebrates, and that teleost genomes carry multiple copies of these co-evolved hormone genes. Sequence comparison showed that each of these genes is highly conserved in different vertebrates and that multiple copies of these peptides in teleosts could be derived from ancient genome duplication and/or lineage-specific intragenic duplications. The present article provides an overview of the calcitonin/intermedin family peptides found in teleost and mammalian genomes, and describes their putative functions. In addition, we demonstrate that one of the intermedin orthologs deduced from the pufferfish (Fugu rubripes) genome shares a conserved signaling activity with mammalian intermedin. The combined results indicate that the physiology associated with each of these family peptides likely evolved during early vertebrate evolution and diverged to serve select physiological functions in different vertebrates.

Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes.

Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders.