Adolfo Rivero-Müller

Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

MicroRNAs-Role in Lung Cancer

Regulation of gene expression is essential for normal physiological functions; thus deregulation of gene expression is common in disease conditions. One level of regulation of gene expression is performed by noncoding RNAs, among which microRNAs (miRNA) are the best studied. Abnormal expression of these molecular players can lead to pathogenic processes such as heart disease, immune system abnormalities, and carcinogenesis, to name but a few. Of a length of 18–25 nucleotides miRNAs are involved in binding partial complementary sequences within the 3′-UTR (3′-untranslated region) of the target mRNAs. Depending on the type of neoplastic transformation, miRNAs can act both as oncogenes (oncomirs) or as tumor suppressors. Because of the great importance of miRNAs, most researches focus on either their role as biomarkers or their potential as therapeutic targets. Herein, we present the review of microRNA biology, function, and tumorigenic potential with emphasis on their role in lung cancer.

Comprehensive Review on Betulin as a Potent Anticancer Agent

Numerous plant-derived substances, and their derivatives, are effective antitumour and chemopreventive agents. Yet, there are also a plethora of tumour types that do not respond, or become resistant, to these natural substances. This requires the discovery of new active compounds. Betulin (BE) is a pentacyclic triterpene and secondary metabolite of plants abundantly found in the outer bark of the birch tree Betulaceae sp. BE displays a broad spectrum of biological and pharmacological properties, among which the anticancer and chemopreventive activity attract most of the attention. In this vein, BE and its natural and synthetic derivatives act specifically on cancer cells with low cytotoxicity towards normal cells. Although the antineoplastic mechanism of action of BE is not well understood yet, several interesting aspects of BEs interactions are coming to light. This review will summarize the anticancer and chemopreventive potential of BE in vitro and in vivo by carefully dissecting and comparing the doses and tumour lines used in previous studies, as well as focusing on mechanisms underlying its activity at cellular and molecular level, and discuss future prospects.

Steroid Receptor Coactivator-3 Is Required for Progesterone Receptor Trans-activation of Target Genes in Response to Gonadotropin-releasing Hormone Treatment of Pituitary Cells

Regulation of gonadotropin production involves interplay between steroids and neuropeptides, and we have examined the effects of gonadotropin-releasing hormones (GnRH I and GnRH II) on progesterone receptor (PR) activation in αT3-1 pituitary cells. Treatment with GnRHs activated a progester-one response element (PRE)-luciferase reporter gene, and this was blocked by protein kinase C and protein kinase A inhibitors but not by RU486. Treatment with GnRHs phosphorylated the PR at Ser294 and increased PR translocation to the nucleus within 1 h. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR-steroid receptor coactivator-3 (SRC-3) interactions within 8 h. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 by the PREs of the luciferase reporter gene or the gonadotopin α-subunit gene promoter was also increased by GnRHs within 8 h, while progesterone-induced recruitment of PR to the PREs occurred in association with much less SRC-3. A small interfering RNA knockdown of type I GnRH receptor levels reduced PR activation by GnRHs, while progesterone-dependent PR activation was unaffected. Moreover, small interfering RNA knockdown of SRC-3 abolished PRE-luciferase trans-activation by the PR in response to GnRHs. Collectively, these data indicate that PR activation by GnRHs in αT3-1 cells is type I GnRH receptor-mediated and that trans-activation of PR-responsive genes requires SRC-3 in this context.

Adrenocortical tumorigenesis, luteinizing hormone receptor and transcription factors GATA-4 and GATA-6

Luteinizing hormone (LH/hCG) responsiveness of normal and pathological human adrenal glands as well as the possibility of constitutive expressions of luteinizing hormone receptor (LHR) in adrenal cortex has been reported. Some recent studies showed a correlation between the LHR and abundant GATA-4 expression in both metastasizing and non-metastasizing human adrenocortical tumors, but not in normal adrenals, implicating the putative relevance of LHR and GATA-4 for adrenocortical pathophysiology. However, the physio- and pathophysiological significance of LHR and GATA-4 in the mechanism of adrenocortical tumorigenesis remains unclear. The paucity of suitable models for adrenal tumorigenesis makes the establishment of proper animal models highly important. LHR expression in the murine adrenal gland is an exception and not found in wild-type (WT) animal. We have previously shown that ectopic LHR expression in the murine adrenal gland can be induced by chronically elevated LH levels. We have generated a gonadotropin-responsive adrenal tumor model in gonadectomized transgenic (TG) mice expressing the inhibin alpha promoter/Simian Virus 40 T antigen transgene (inhalpha/Tag). Given the induction of expression and regulation of GATA-4 and GATA-6 zinc finger transcription factors in the gonads by gonadotropins, this review will explore their relationship to LHR expression and their role in adrenocortical tumorigenesis. A functional link between LHR and GATA-4 actions in the adrenal pathophysiology is proposed.

Di/Oligomerization of GPCRs—Mechanisms and Functional Significance

Initially considered as strict and rigid monomers, GPCRs have shown an ever-increasing flexibility in the number of intermolecular interactions they can participate in. Few rules can be applied to all members of this receptor superfamily, as the functional roles of resulting dimers or oligomers vary widely from biosynthesis, functional complementation, enhancement and control of signaling, positive and negative cooperativity, signal desensitization, membrane trafficking, and pharmacological diversity, among others. Receptor dimerization/oligomerization is a challenging phenomenon to disentangle even in vitro and thus in vivo evidence for its functional significance requires employment of a variety of cutting-edge techniques. Nevertheless, the evidence for GPCR di/oligomerization at the physiological level is mounting and has begun to shed light on an intricate network of interactions that challenge the previous knowledge on how this family of receptors function. Here, we summarize the evidence for GPCR di/oligomerization in living organisms with a particular focus of their involvement in health and disease.

Enhanced LH action in transgenic female mice expressing hCGβ-subunit induces pituitary prolactinomas; the role of high progesterone levels

The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) β-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P4) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P4 levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P4 treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P4 was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P4. If extrapolated to humans, and given the importance of endogenous P4 and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas.

Transgenic GATA-4 expression induces adrenocortical tumorigenesis in C57Bl/6 mice.

Summary A link between elevated luteinizing hormone (LH) levels, GATA-4 and LH receptor (LHCGR) expression and gonadotropin-dependent adrenocortical tumorigenesis in humans and mice has been shown. To assess the mechanistic tumorigenic interrelationships between these factors, we transgenically expressed Gata4 under the 21-hydroxylase promoter (Cyp21a1, 21-OH) in C57Bl/6N mice. There was a gradual age-dependent increase of GATA-4 expression only in 21-OH-GATA-4 (TG) female adrenals, in association with slowly progressing neoplasia of non-steroidogenic spindle-shaped A cells in the subcapsular cortex. Gonadectomy (GDX), apparently through direct action of elevated serum LH, markedly enhanced the adrenocortical neoplasia, which now also appeared in GDX TG males. The neoplastic areas of the post-GDX TG adrenals contained, besides A cells, larger lipid-laden, steroidogenically active and LHCGR-positive B cells. Prolonged (>10 months) exposure to elevated post-GDX LH levels resulted in formation of adrenocortical adenomas in the TG mice. Intact and GDX TG mouse adrenals displayed elevated FOG-2 and decreased GATA-6 expression. Additionally, increased expression/activation of components of the Inhbb–Acvr2a–Acvr1c–Smad2/3 signaling system was observed in 12-month-old GDX TG adrenals. Our findings show that two distinct GATA-4-dependent populations of neoplastic adrenocortical cells form: non-steroidogenic LH-independent A cells and steroidogenic LH-dependent B cells.

Keratin 8-deletion induced colitis predisposes to murine colorectal cancer enforced by the inflammasome and IL-22 pathway

Keratins (K) are intermediate filament proteins important in protection from cellular stress. K8, K18 and K19 are the main components of keratin filaments in colonic epithelia but their role in intestinal diseases remains ambiguous. A function for keratins in intestinal health is supported by the K8-knock-out (K8(-/-)) mouse which manifests an early chronic ulcerative colitis-like inflammatory bowel disease and epithelial hyperproliferation. We tested whether K8(-/-) mice are more susceptible to colorectal cancer (CRC) compared to K8 wild type (K8(+/+)), and K8 heterozygote (K8(+/-)) mice showing increased proliferation but no inflammation. K8(-/-) mice did not develop CRC spontaneously, but had dramatically increased numbers of tumors in the distal colon in the azoxymethane (AOM) and Apc(Min/+) CRC models while neither K8(+/+) nor K8(+/-) mice were susceptible. Upregulation of IL-22 in combination with a complete loss of its negative regulator IL-22BP, and increased downstream STAT3-signaling in K8(-/-) and K8(-/-)Apc(Min/+) colonic epithelia confirmed that the IL-22 pathway, important in inflammation, proliferation and tissue regeneration, was activated. The nearly total loss of IL-22BP correlated with an activated inflammasome leading to increased cleaved caspase-1, and the putative IL-22BP inhibitor, IL-18, as well as a decrease in ALDH1/2. Ablation of K8 in a colorectal cancer cell line similarly resulted in increased IL-18 and decreased ALDH1/2. K8/K18 co-immunoprecipitated with pro-caspase-1, a component of the inflammasome in the colon, which suggests that keratins modulate inflammasome activity and protect the colon from inflammation and tumorigenesis. The K8-null mouse models also provide novel epithelial-derived robust colon-specific CRC models.

Reconstruction of Mouse Testicular Cellular Microenvironments in Long-Term Seminiferous Tubule Culture

Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilms tumour antigen 1 (WT1) and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA) and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger), LIN28, Gpr125 (G protein-coupled receptor 125), CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs) that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2–4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to establish in vitro conditions that allow spontaneous reconstruction of testicular cellular microenvironments.