Expressão gênica do receptor do hormônio luteinizante (LHR), em células da teca e da granulosa de folículos antrais bovinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão do gene da leptina e seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras

Objetivou-se com este trabalho avaliar os efeitos de uma dieta de alto nível de energia e proteína combinada com a aplicação de bST no perfil de expressão dos genes da leptina e de seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras. Foram utilizadas amostras de parênquima mamário de 32 novilhas holandesas distribuídas aleatoriamente em quatro tratamentos (n=8): dieta com alto ou baixo teor de energia e proteína combinada ou não com a aplicação de bST. O delineamento utilizado foi em blocos casualizados com arranjo de tratamentos em esquema fatorial 2 × 2. A extração do RNA total das amostras de tecido foi feita e o nível de expressão gênica foi analisado por qRT-PCR utilizando-se o gene da glicuronidase β como controle, pelo método 2-ΔΔCt. Animais que receberam a dieta com alto conteúdo de energia e proteína apresentaram maior expressão de mRNA de leptina, com aumento de 56%, e menor expressão de mRNA do receptor Ob-Rb, com redução de 18%. Por outro lado, a aplicação de bST resultou em diminuição da expressão do mRNA de leptina e do receptor Ob-Rb em 74% e 23%, respectivamente. Não houve interação entre dieta e aplicação de bST. O aumento na expressão de leptina pode explicar, ao menos em parte, os efeitos negativos da dieta de alta energia e proteína, oferecida no período pré-púbere, sobre a produção de leite de novilhas leiteiras.

Evidence of the Importance of the First Intracellular Loop of Prokineticin Receptor 2 in Receptor Function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2. (Molecular Endocrinology 26: 1417-1427, 2012)

Parathyroid hormone and phosphorus overload in uremia: impact on cardiovascular system

Background. Cardiac remodeling in uremia is characterized by left ventricular hypertrophy, interstitial fibrosis and microvascular disease. Cardiovascular disease is the leading cause of death in uremic patients, but coronary events alone are not the prevalent cause, sudden death and heart failure are. We studied the cardiac remodeling in experimental uremia, evaluating the isolated effect of parathyroid hormone (PTH) and phosphorus. Methods. Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx); they also received vehicle (V) and PTH at normal (nPTH) or high (hPTH) doses. They were fed with a poor-phosphorus (pP) or rich-phosphorus (rP) diet and were divided into the following groups: 'Sham': G1 (V + normal-phosphorus diet (np)) and 'Nx + PTx': G2 (nPTH + pP), G3 (nPTH + rP), G4 (hPTH + pP) and G5 (hPTH + rP). After 8 weeks, biochemical analysis, myocardium morphometry and arteriolar morphological analysis were performed. In addition, using immunohistochemical analysis, we evaluated angiotensin II, alpha-actin, transforming growth factor-beta (TGF-beta) and nitrotyrosine, as well as fibroblast growth factor-23 (FGF-23), fibroblast growth factor receptor-1 (FGFR-1) and runt-related transcription factor-2 (Runx-2) expression. Results. Nx animals presented higher serum creatinine levels as well as arterial hypertension. Higher PTH levels were associated with myocardial hypertrophy and fibrosis as well as a higher coronary lesion score. High PTH animals also presented a higher myocardial expression of TGF-beta, angiotensin II, FGF-23 and nitrotyrosine and a lower expression of alpha-actin. Phosphorus overload was associated with higher serum FGF-23 levels and Runx-2, as well as myocardial hypertrophy. FGFR-1 was positive in the cardiomyocytes of all groups as well as in calcified coronaries of G4 and G5 whereas Runx-2 was positive in G3, G4 and G5. Conclusion. In uremia, PTH and phosphorus overload are both independently associated with major changes related to the cardiac remodeling process, emphasizing the need for a better control of these factors in chronic kidney disease.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

An open-label, noncomparative phase II trial to evaluate the efficacy and safety of docetaxel in combination with gefitinib in patients with hormone-refractory metastatic prostate cancer

BACKGROUND: Prostate cancer is the most common type of cancer in men, however, therapeutic options are limited. 50-90% of hormone-refractory prostate cancer cells show an overexpression of epidermal growth factor receptor (EGFR), which may contribute to uncontrolled proliferation and resistance to chemotherapy. In vitro, gefitinib, an orally administered tyrosine kinase inhibitor, has shown a significant increase in antitumor activity when combined with chemotherapy. PATIENTS AND METHODS: In this phase II study, the safety and efficacy of gefitinib in combination with docetaxel, a chemotherapeutic agent commonly used for prostate cancer, was investigated in patients with hormone-refractory prostate cancer (HRPC). 37 patients with HRPC were treated continuously with gefitinib 250 mg once daily and docetaxel 35 mg/m2 i.v. for up to 6 cycles. PSA response, defined as a =50% decrease in serum PSA compared with trial entry, was the primary efficacy parameter. PSA levels were measured at prescribed intervals. RESULTS: The response rate and duration of response were consistent with those seen with docetaxel monotherapy. The combination of docetaxel and gefitinib was reasonably well tolerated in this study. CONCLUSION: Future studies should investigate whether patients with specific tumor characteristics, e.g. EGFR protein overexpression, respond better to gefitinib than patients without, leading to a more customized therapy option.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b−/− males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b−/− mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b−/− mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Effects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancer

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPARγ is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation of this receptor with specific ligands exerts an inhibitory effect on the growth of prostate cancer cell lines. Further, we show that prostate cancer and cell lines do not have intragenic mutations in the PPARγ gene, although 40% of the informative tumors have hemizygous deletions of this gene. Based on our preclinical data, we conducted a phase II clinical study in patients with advanced prostate cancer using troglitazone, a PPARγ ligand used for the treatment of type 2 diabetes. Forty-one men with histologically confirmed prostate cancer and no symptomatic metastatic disease were treated orally with troglitazone. An unexpectedly high incidence of prolonged stabilization of prostate-specific antigen was seen in patients treated with troglitazone. In addition, one patient had a dramatic decrease in serum prostate-specific antigen to nearly undetectable levels. These data suggest that PPARγ may serve as a biological modifier in human prostate cancer and its therapeutic potential in this disease should be further investigated.

Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 μg/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054,522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.

The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling

The extracellular domain of p185c-neu can be viewed as a complex structure of four subdomains, two of which are cysteine-rich subdomains. We have investigated the contribution of these distinct p185c-neu extracellular subdomains to p185/epidermal growth factor receptor (EGFR) heteromer formation and EGF-induced heteromeric signaling. Our studies indicate that at least two separate p185 subdomains, a region spanning subdomains I and II and subdomain IV are involved in association of p185 with the EGFR. We also demonstrated that subdomain IV reduced the heteromeric signaling and transforming activities induced by EGF after associating with EGFR. When 126 aa were deleted from subdomain IV, this small subdomain IV-derived fragment could still lead to heterodimers with EGFR and suppress EGF-induced mitogen-activated protein kinase activation and subsequent transformation abilities. These data provide information about trans-inhibitory mechanisms of mutant p185 species and also indicate that both the entire and a part of subdomain IV may represent a therapeutic target for erbB-overexpressing tumors. Finally, these studies define a basic feature of receptor-receptor associations that are determined by cystine-knot containing subdomains.

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.