Dimitris K. Grammatopoulos

Functional characteristics of CRH receptors and potential clinical applications of CRH-receptor antagonists

Corticotropin-releasing hormone (CRH) plays a major role in coordinating the behavioral, endocrine, autonomic and immune responses to stress. CRH and CRH-related peptides and their receptors are present in the central nervous system and in a wide variety of peripheral tissues, including the immune, cardiovascular and reproductive systems, and have been associated with the pathophysiology of many disease states. These observations have led to the development of several CRH receptor type-selective antagonists, which have been used experimentally to elucidate the role of CRH and related peptides in physiological and disease processes, such as anxiety and depression, sleep disorders, addictive behavior, inflammatory and allergic disorders, neurological diseases and pre-term labor. Because of the complex network of multiple CRH receptor subtypes and their tissue- and agonist-specific signaling diversity, antagonists need to be developed that can target specific CRH receptor isoform-driven signaling pathways.

Role of corticotropin-releasing hormone in onset of labour

Corticotropin-releasing hormone (CRH) derived from the placenta is secreted into the maternal circulation in large amounts during the third trimester of human pregnancy and may have an important role in the onset of labour. Although the biological role of CRH remains enigmatic, the presence of functional CRH receptors in the myometrium suggests that CRH may modulate myometrial contractility and hence parturition. CRH action is mediated via multiple receptor subtypes and pregnancy results in differential receptor expression. These receptors are primarily linked to the adenylate cyclase second messenger system, which would help the intracellular microenvironment to maintain the required myometrial quiescence. CRH can exert further actions such as inhibition of prostaglandin production to prevent contractions. At term under the influence of oxytocin there is a modification in the coupling mechanisms that leads to a decrease in the biological activity of the CRH receptor and in the generation of cyclic adenosine monophosphate which favours myometrial contractions. CRH, via distinct receptor subtypes, is then able to enhance the contractile response of the myometrium. This hypothesis places CRH in a central role in coordinating the smooth transition from a state of relaxation to one of contraction.

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins

The wide distribution of corticotrophin‐releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor‐mediated guanine‐nucleotide binding protein (G‐proteins) activation, we recently demonstrated that expressed type 1α CRH receptors are capable of activating multiple G‐proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G‐proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [α‐32P]‐GTP‐γ‐azidoanilide to various G‐protein α‐chains. Our results show that CRH receptors are coupled to and activate at least five different G‐proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα‐protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.

Potential signalling pathways underlying corticotrophin‐releasing hormone‐mediated neuroprotection from excitotoxicity in rat hippocampus

In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of corticotrophin‐releasing hormone (CRH), the 41‐amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2 pm), prevented glutamate‐induced neurotoxicity via receptor‐mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate‐induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphorylation mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuroprotection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult.

The identification of mitochondrial DNA variants in glioblastoma multiforme

BackgroundMitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis.ResultsUsing next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas.ConclusionsThese novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.

Insights into mechanisms of corticotropin-releasing hormone receptor signal transduction.

During evolution, mammals have developed remarkably similar molecular mechanisms to respond to external challenges and maintain survival. Critical regulators of these mechanisms are the family of ‘stress’‐peptides that consists of the corticotropin‐releasing hormone (CRH) and urocortins (Ucns). These neuropeptides ‘fine‐tune’ integration of an intricate series of physiological responses involving the autonomic, endocrine, immune, cardiovascular and reproductive systems, which induce a spectrum of behavioural and homeostatic changes. CRH and Ucns exert their actions by activating two types of CRH receptors (CRH‐R), CRH‐R1 and CRH‐R2, which belong to the class‐B1 family of GPCRs. The CRH‐Rs exhibit signalling promiscuity facilitated by their ability to couple to multiple G‐proteins and regulate diverse intracellular networks that involve intracellular effectors such as cAMP and an array of PKs in an agonist and tissue‐specific manner, a property that allows them to exert unique roles in the integration of homeostatic mechanisms. We only now begin to unravel the plethora of CRH‐R biological actions and the transcriptional and post‐translational mechanisms such as alternative mRNA splicing or phosphorylation‐mediated desensitization developed to tightly control CRH‐Rs biological activity and regulate their physiological actions. This review summarizes the current understanding of CRH‐R signalling complexity and regulatory mechanisms that underpin cellular responses to CRH and Ucns.

Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells

The biological actions of corticotropin-releasing hormone (CRH) in the human myometrium during pregnancy and labor are unknown. We hypothesized that CRH may modulate the nitric oxide system, and influence myometrial relaxation/contractility. Incubation of myometrial cells with CRH, but not urocortin II or urocortin III, for 8–16 h significantly induced mRNA and protein expression of endothelial and brain but not inducible nitric oxide synthase (NOS) isoforms. This action resulted in increased activity of soluble guanylate cyclase (GCs), demonstrated by the enhanced cGMP-producing capacity of the NO donor, sodium nitroprusside. CRH also caused acute activation of the membrane-bound GC, shown by increased basal or atrial natriuretic peptide (ANP)-stimulated cGMP production. These effects appeared to be mediated via the R1 receptors because the CRH receptor antagonists, astressin and antalarmin but not anti-sauvagine 30, could block them. The acute effects of CRH were significantly reduced by inhibition of protein kinase A (PKA) activity, suggesting it is partially PKA dependant. Activation of protein kinase C (PKC) resulted in significant inhibition of both ANP-and CRH-stimulated cGMP production, suggesting a direct effect of PKC on membrane-bound GC. In conclusion, CRH appears to have a dual effect on myometrial NOS/GC pathway, a short term effect predominantly mediated by PKA, and a long-term effect increasing constitutive NOS expression, mediated by a PKA-independent mechanism. This mechanism could potentially be active during human pregnancy, and, because cGMP stimulates myometrial relaxation, these findings further suggest that during pregnancy CRH primarily activates intracellular signals that contribute to the maintenance of myometrial quiescence.

Interplay of cAMP and MAPK pathways in hCG secretion and fusogenic gene expression in a trophoblast cell line.

Differentiation of human placental mononuclear trophoblasts into a multinucleate syncytium involves up-regulation of key proteins promoting cell fusion and increased capacity for placental hormonogenesis. It is well established that the activation of adenylyl cyclase leads to increased expression of trophoblast fusogenic gene machinery and human chorionic gonadotropin (hCG) secretion. We used the forskolin-induced syncytialisation of BeWo choriocarcinoma cells as a model to characterise in detail the signalling pathway downstream of adenylyl cyclase. Forskolin treatment induced a rapid and potent ERK1/2 and p38MAPK phosphorylation; this cascade required PKA-AKAP interactions and led to downstream CREB-1/ATF-1 phosphorylation via ERK1/2-dependent but p38MAPK-independent mechanisms. Interestingly both p38MAPK and ERK1/2 were involved in forskolin-induced hCG-secretion, suggesting the presence of additional p38MAPK-dependent but CREB-1/ATF-1-independent pathways. Forskolin treatment of BeWo cells significantly up-regulated the expression of various fusogenic gene mRNAs, including syncytin-1 and -2 (by 3- and 10-fold, respectively) the transcription factors old astrocyte specifically induced substance (OASIS) and glial cells missing a (GCMa) (by 3- and 6-fold, respectively) and the syncytin-2 receptor, major facilitator superfamily domain containing 2 (MFSD2) (by 2-fold). Up-regulation of AKAP79 and AKAP250 (by 2.5- and 4-fold, respectively) was also identified in forskolin-treated BeWo cells. Forskolin effects on all these genes were suppressed by chemical inhibition of p38MAPK whereas only specific genes were sensitive to ERK1/2 inhibition. This data provide novel insights into the signalling molecules and mechanisms regulating fusogenic gene expression by the adenylyl cyclase pathway.

Placental Corticotrophin-Releasing Hormone and its Receptors in Human Pregnancy and Labour: Still a Scientific Enigma

It is now accepted that, in humans, placental corticotrophin‐releasing hormone (CRH) is involved in the mechanisms controlling the onset of labour; however, the precise biological role in foeto–maternal tissues remain enigmatic. Maternal plasma levels of CRH rise exponentially as pregnancy progresses towards term and peak during labour; however, evidence to link this with an active role in the onset and progression of labour, is still inconclusive. Certainly, one of the tissues targeted by CRH is the myometrial smooth muscle, which expresses a plethora of specific CRH receptors. This finding implicates CRH in the mechanisms preparing the myometrial microenvironment for the onset of labour and possibly in the regulation of active contractility during labour. Other gestational tissues also targeted by CRH include the placenta, foetal membranes and foetal adrenals, where CRH might regulate distinct physiological functions, ranging from control of vascular tone to adrenal steroidogenesis and prostaglandin synthesis and activity. Given the unique, among mammals, pattern of human placental CRH secretion and CRH receptor expression and signalling during pregnancy and labour, there are only limited biological tools available to delineate the actions of CRH in foeto–maternal tissues, primarily based on in vitro characterisation of the signalling and molecular events driven by CRH. This review will set in context the current concepts about the role of CRH and its receptors during pregnancy and labour, focusing on the unresolved questions and paradoxes that currently exist.

Measurement of thyroglobulin mRNA in peripheral blood as an adjunctive test for monitoring thyroid cancer

Aims: Monitoring treated patients with thyroid cancer for recurrent or metastatic disease is currently based upon the serial measurement of circulating plasma thyroglobulin (Tg) concentrations. However, the clinical usefulness of Tg immunoassays is limited by poor sensitivity and interference from anti-Tg antibodies. This study investigated whether the detection of Tg mRNA in peripheral blood, using reverse transcriptase polymerase chain reaction (RT-PCR), is of value in the biochemical surveillance of patients with thyroid cancer. Methods: RNA was extracted from peripheral blood of five normal controls, six patients with abnormal thyroid function tests, and 28 patients who had undergone thyroidectomy for well differentiated thyroid cancer. From each, an 87 bp product from base pair 262 to 348 in the cDNA sequence of the thyroglobulin gene was amplified by RT-PCR. Results: Tg mRNA was detected in normal individuals and patients with thyroid cancer. In the group of patients studied, identification of metastatic thyroid tissue by radioiodine scanning correlated better with Tg mRNA assay results than with serum Tg concentrations (accuracy 84% v 75%). No interference from circulating Tg antibodies was apparent. In patients studied prospectively over a 12 month period, there was a significant correlation between detectable Tg mRNA in peripheral blood and the presence or absence of metastatic disease, as demonstrated by radioiodine scanning. Conclusions: These results suggest that detection of Tg mRNA in blood is a more sensitive marker for metastatic thyroid disease than Tg immunoassay, and appears to be unaffected by the presence of circulating anti-Tg antibodies.