CRH (Corticotropin-Releasing Hormone) and related peptides play a major role in coordinating the behavioral, endocrine, cardiovascular, autonomic and immune mechanisms that allow mammals to adapt under both basal and stressful conditions. Their actions are mediated through activation of two types of GPCRs (G-Protein-Coupled Receptors): CRHR1 (CRH Receptor-1) and CRHR2 (CRH Receptor-2) encoded by separate genes (Ref.1). CRH and CRH-related peptides and their receptors are widely distributed in the central nervous system, and in a variety of peripheral tissues, including the immune, cardiovascular and reproductive systems, adrenals, lungs, liver, stomach, pancreas, small intestine, skin tissues, and also in some types of human tumors. The ubiquitous distribution of CRH-related peptides and CRHRs, capable of activating diverse signaling mechanisms in different tissues, gives this system enormous versatility and plasticity (Ref.2).
CRH, a 41-amino acid neuropeptide is secreted by the paraventricular nucleus of the hypothalamus (also synthesized in human trophoblast, amnion, chorion, decidua, and myometrium in small amounts). As its name indicates, CRH is the physiological stimulus of POMC (Pro-opiomelanocortin) transcription and corticotrophin, i.e., ACTH (Adrenocorticotropic Hormone) secretion in cells in the anterior lobe of the pituitary and it acts as a major mediator of the stress response. ACTH in turn controls the secretion of steroid hormones by the adrenal cortex, which affects glucose, protein, and fat metabolism. While CRH accounts for a large part of the corticotropin-releasing activity of extracts in the hypothalamus, it does not account for all of it. Structurally related peptides, including the mammalian peptides: Ucn (Urocortin), Ucn2 (Urocortin-2) and Ucn3 (Urocortin-3) which are differentially distributed in the brain and periphery, and appear to be involved in an array of physiological processes, such as ingestive behavior, inflammation, lipogenesis and vascularization mediated through the activation of CRHRs (Ref.1). CRHRs represent a family with at least two distinct members in humans CRHR1 and CRHR2, encoded by separate genes, sharing high sequence homology (70%), and differing markedly in their pharmacological profiles and anatomical distribution. These receptors are seven transmembrane receptor proteins coupled to the G-protein signaling system and belong to the class-II GPCR superfamily (Ref.3). CRHR1 is a 415-amino acid protein, containing seven hydrophobic helices that are predicted to span the plasma membrane; This widespread both within the central nervous system and its periphery; and shows high affinity for CRH and Ucn. In contrast, CRHR2 receptors show a clear preference in their affinity for Ucn-like ligands. CRHR1 has been implicated in mediating normal responses to stress, whereas CRHR2 seems to be involved in fine tuning stress responses (Ref.4 & 5).
Signals from CRH and CRH-related peptides are transduced across cell membranes via activation of both the CRHRs (Ref.1 & 4). In many tissues (e.g. brain, heart, myometrium), stimulation of either type of CRHR leads to the activation of AC (Adenylyl Cyclase) and the subsequent increase in cAMP levels. However, in certain tissues (i.e. testes, placenta), CRH is unable to activate the AC pathway, whereas it can stimulate alternative signalling cascades, such as stimulation of Phosphoinositol hydrolysis. Such differential outcome of CRHR activation has been attributed to multiple G-Protein activation, a finding that predicts activation of several different second messenger signals and suggests that CRH and CRH-related peptides can generate various responses in different target tissues (Ref.3). CRHRs modulate a plethora of intracellular protein kinases, such as PKA, PKC, PKB (Protein Kinase-B)/Akt, the ERKs (Extracellular-signal Regulated Kinases) and p38MAPKs (Mitogen-Activated Protein Kinases), as well as other important signaling intermediates, such as Ca2+, NOS (Nitric Oxide Synthase), GC (Guanylyl Cyclase), Steroidogenic enzymes, Prostaglandins, FasL(Fas Ligand) etc. in a tissue-specific manner. These proteins in turn stimulate various transcription factors like c-Jun, c-Fos, JunD, Elk1, MEF2 (MADS Box Transcription Enhancer Factor-2), and CREB which act directly on the POMC promoter and stimulate ACTH biosynthesis along with the expression of several other genes (Ref.6 & 7).
Agonist binding causes a change in the structural conformation of these receptors leading to signal transduction across the cell membrane through activation of heterotrimeric G-proteins. In most tissues, stimulation of either CRHR1 or CRHR2 by CRH and CRH-related peptides triggers activation of AC with the production of cAMP and subsequent activation of PKA (Protein Kinase-A)-dependent pathways. Activation of AC is facilitated by G-AlphaS, whereas, it is inhibited by G-AlphaI. PKA activation by CRH and cAMP activates two main transduction pathways: one of these depends on calcium entry at the plasmatic membrane through the activation of CaCn (Calcium Channel) and involves CAMKII activity; the other pathway is calcium independent. Activated PKA phosphorylates CREB, which initiates expression of several genes. The MAPK pathway is also activated by CRH and cAMP by Calcium-dependent and -independent mechanisms and involves Rap1, BRaf, MEK, and ERK activities. PLC (Phospholipase-C) is also activated by CRHR in a G-AlphaQ-dependent manner with the production of DAG (Diacylglycerol) and IP3 (Inositol Triphosphate), which in turn activate PKA, PKC (Protein Kinase-C)-dependent and Calcium-activated pathways respectively (Ref.4). IP3 stimulates Ca2+ release from the ER (Endoplasmic Reticulum) through its action on the IP3R (IP3 Receptor). Activation of PKC pathways assumes a central position in CRHRsignaling. PKC pathway also mediates several of the immune effects of CRH (Ref.8). CRH stimulates human fetal adrenal steroidogenesis and prostaglandin synthesis by human placenta via the PKC pathway by the activation of NOS, Nitric Oxide, GC and cGMP (cyclic Guanosine 3, 5-Cyclic Monophosphate). Activation of Nitric Oxide by CRH brings about vasodilation. Similarly, the effects of CRH on Leydig, human myometrial, and hippocampal cells also involve activation of the PKC pathway by activating the MAPKs. PKA, PKC activates MAPKKKs (Mitogen-Activated Protein Kinase Kinase Kinases) through Raf1 activation. MAPKKKs activate the ERKs (ERK1 and ERK2) and p38 (Ref.9).
The MAPK pathway is also activated by CRH and cAMP by calcium-dependent and -independent mechanisms and involves Rap1, BRaf, MEK1 (MAPK/ERK Kinase-1), and
ERK activities. These different transduction pathways regulate ACTH induction by different mechanisms: 1) calcium-dependent and -independent induction of Nur77, which does not involve MAPK activity; and 2) a MAPK stimulation of transcription and activity of Nur77 (Ref.10). CRHR-mediated activation of MAPK signal transduction pathways are observed in several cell types, including normal and neoplastic corticotropes, cardiac myocytes, and pregnant uterine myocyte cells and are involved in diversified tissue-specific responses (Ref.4). For example, in neuronal and hippocampal cells, the
CRHR- MAPK cascade mediates the neuroprotective effects of the CRH and CRH-like peptides. FasL activation by CRHR-activated p38 MAPKs culminates in apoptosis (Ref.11).
CRHRs located in the heart and vasculature mediate the ionotropic, vasodilatory, and anti-edema effects of CRH and/or CRH-related peptides. In immune cells, the local effects of CRH are complex since it has been reported to both inhibit and stimulate production of the proinflammatory cytokines (IL-1 and IL-6) by peripheral blood mononuclear cells. CRHR induces the transcription of the COX2 (Cyclooxygenase-2) gene in human fetal membranes through the involvement of of a GAlphaQ-PKC pathway. CRHR activation stimulates prostaglandin production in human fetal membranes and placenta and induces vasodilation in the human fetal-placental circulation via a NO (Nitric Oxide)-cGMP-mediated pathway. Activation of PKC also has a role in stimulating the release of arachidonic acid and Prostaglandins in human amnion cells (Ref.5).
CREB phosphorylation by CRHR-activated PKA leads to activation of BDNF (Brain-Derived Neurotrophic Factor) expression. However, concurrent stimulation of CB1 (Cannabinoid Receptor-1) with its agonist: WIN 55212-2 (2,3-Dihydro-5-methyl-3-((4-morpholinyl)methyl) pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate) inhibits CRH- and depolarization-induced BDNF expression through its negative effect on the cAMP signaling cascade and its ability to inhibit the activity of Ca2+ channels (Ref.12). CRHR signal transduction pathway regulates keratinocyte differentiation and proliferation in an organized and sequential manner. Binding of CRH to the CRHR results in the activation of PKC, which stimulates Jun-Fos binding activity. c-Jun and c-Fos stimulate p16 resulting in G0/1 arrest (cell cycle arrest), and JunD, also a member of Activator Protein-1 complexes) induces keratinocyte differentiation program. The cell cycle withdrawal is associated with the induction of keratinocyte differentiation. Thus, CRH stimulates the expression of Cytokeratin1 and Involucrin, on both mRNA and protein levels. CRHR signal transduction pathway regulates keratinocyte differentiation and proliferation in an organized and sequential manner. Binding of CRH to the CRHR results in the activation of PKC, which stimulates Jun-Fos binding activity. c-Jun and c-Fos stimulate p16 resulting in G0/1 arrest (cell cycle arrest), and JunD, also a member of Activator Protein-1 complexes) induces keratinocyte differentiation program. The cell cycle withdrawal is associated with the induction of keratinocyte differentiation. Thus, CRH stimulates the expression of Cytokeratin1 and Involucrin, on both mRNA and protein levels (Ref.13). CRH has proinflammatory effects in mast cells. It induces VEGF release in human mast cells via selective activation of the cAMP--> PKA-->p38MAPK signaling pathway (Ref.9).
ACTH secretion by CRHR pathway has been found to be augmented by its crosstalk with the Shh (Sonic Hedgehog) pathway. Shh is a signaling protein that binds to Ptc (Patched) and mediates its effects through Gli transcription factors and is important in regulating survival and growth in both the embryo and the adult. Shh binds to its receptor Ptc, that subsequently activates the Smo (Smoothened) receptor, leading to activation of the Gli transcription factors. The signal is mediated by Gli2 and Gli3, which both activate Gli1, which in turn is responsible for the activation of the downstream target genes. Shh pathway and, in particular, the Gli1 transcription factor play a role in the expression of CRHR1, intracellular cAMP, and regulation of ACTH in response to CRH. CRH also increases the Gli-dependent transcription. Thus, Shh and CRH have additive effects on ACTH production resulting from a crosstalk between the respective signal transduction pathways at different levels (Ref.14).
CRH and CRH-related peptides, and their receptors, form an important physiological system, influencing a wide spectrum of behavioral, cardiovascular, metabolic and immune mechanisms that allow mammals to coordinate the adaptive behavioral and physical changes that occur during stress. However, at high levels, for example when hypersecreted chronically, CRH causes anxiety, sleep disruption and adverse changes in cardiovascular, metabolic and immune functions (Ref.2 & 6). Furthermore, CRH has been implicated in the pathophysiology of various psychiatric, neuroendocrine and neurological illnesses; in particular, there is evidence linking abnormalities in the CRH system to chronic anxiety disorder, melancholic and atypical depression, chronic pain and fatigue states, sleep disorders, addictive behavior, acute and chronic neurodegeneration, allergic and autoimmune inflammatory disorders, the metabolic syndrome, gastrointestinal diseases and pre-term labor (Ref.15).