Peripheral corticotropin-releasing hormone and urocortin in the control of the immune response

Immunological and cellular stress signals trigger the release of corticotropin-releasing hormone (CRH) from the spleen, thymus and inflamed tissue. In vivo and in vitro studies generally suggest that peripheral, immune CRH has pro-inflammatory effects and acts in a paracrine manner by binding to CRH-R1 and CRH-R2 receptors on neighboring immune cells. However, it now seems likely that some of the suggested pro-inflammatory actions of CRH may be attributed to novel CRH-like peptides or to the related peptide, urocortin, which is also present in immune cells and has especially high affinity for CRH-R2 receptors.     

Inhibition of 5alpha-reductase activity in SZ95 sebocytes and HaCaT keratinocytes in vitro.

Inhibition of 5alpha-reductase type 1 has been considered to be a promising target for treatment of androgen-dependent skin disorders, however, currently published clinical results on acne treatment are rather disappointing. In this study, the influence of selective inhibitors of 5alpha-reductase on testosterone metabolism within SZ95 sebocytes and HaCaT keratinocytes IN VITRO was investigated. In both cell types, the isotype 1 inhibitor MK386 completely inhibited the conversion of testosterone to 5alpha-dihydrotestosterone in concentrations higher than 10 (-9) M. Inhibitors of the isotype 2 such as finasteride, dihydrofinasteride, and turosteride, were >100-fold less active, while, as expected, androgen receptor inhibitors did not affect the 5alpha-reductase activity. MK386, but not finasteride, reduced testosterone-stimulated proliferation and slightly reduced the testosterone-induced increase in the amount of SZ95 sebocyte proteins. The androgen receptor inhibitor cyproterone acetate exhibited no effect on testosterone-induced proliferation, but inhibited the 5alpha-dihydrotestosterone-induced sebocyte proliferation. Our experimental findings and the existing clinical results indicate that the inhibition of 5alpha-reductase activity alone may be insufficient to reduce overall sebocyte activity and improve acne lesions.     

CRH functions as a growth factor/cytokine in the skin

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.     

A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes

CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.     

Corticotropin releasing hormone and related peptides can act as bioregulatory factors in human keratinocytes

Summary Following previous findings in human skin of the functional expression of genes for the corticotropin releasing hormone (CHR) receptor type 1 (CRH-R1) and CRH itself, we searched for local phenotypic effects for peptides related to CRH. We now report that CRH, sauvagine, and urocortin inhibit proliferation of human HaCaT keratinocytes in a dose-dependent manner. The peptides produced variable cyclic adenosine 3′∶5′-monophosphate stimulation with CRH having the highest potency. Binding of iodine 125 CRH to intact keratinocytes was inhibited by increasing doses of CRH, sauvagine, or urocortin, all showing equal inhibitory potency. Immunocytochemistry identified CRH-R1 immunoreactivity in HaCaT keratinocytes. In conclusion, CRH (exogenous or produced locally) and the related urocortin and sauvagine peptides can modify human keratinocyte phenotype through a receptor-mediated pathway.     

Characterization of the vitamin D endocrine system in human sebocytes in vitro

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25OHase), 25-hydroxyvitamin D-1α-hydroxylase (1αOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1αOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)₂D₃ resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1αOHase were detected, our findings indicate that the full length product represents the major 1αOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)₂D₃ synthesis/metabolism.     

Pleiotropic effects of corticotropin releasing hormone on normal human skin keratinocytes

The hypothalamic-pituitary-adrenal (HPA) axis is the major stress response system. Several components of the HPA axis, such as corticotropin-releasing hormone (CRH) and POMC peptides and their receptors are also present in the skin. In earlier studies, we showed that CRH inhibits cellular proliferation of immortalized human keratinocytes. We now examine further the functional activity of the HPA axis in the skin, by characterizing the actions of CRH on normal foreskin keratinocytes. The CRH receptor was detected as CRH-R1 antigen at 47 kDa in the cultured keratinocytes by Western blotting, and immunohistochemistry demonstrated its presence in the epidermal and follicular keratinocytes. CRH is also biologically active in cultured keratinocytes, where it inhibits proliferation and enhances the interferon-gamma-stimulated expression of the hCAM and ICAM-1 adhesion molecules and of the HLA-DR antigen. These effects were concentration-dependent, with maximal activity at CRH 10(-7) M. Thus, in the keratinocyte, the most important cellular component of the epidermis, CRH appears to induce a shift in energy metabolism away from proliferation activity, and toward the enhancement of immunoactivity. Therefore, similar to its central actions, cutaneous CRH may also he involved in the stress response, but at a highly localized level.     

Corticotropin releasing hormone stimulates proliferation of keratinocytes

Corticotropin releasing hormone (CRH) is a potent mediator of stress responses and stress-induced disorders. Consistent with the broad range of roles proposed for CRH, high-affinity binding sites have been found in various peripheral sites. Recently two types of CRH specific receptor have been identified. Expression of CRH receptor 1 (CRH-R1) gene has been detected in human keratinocyte, but the effects of CRH to keratinocytes are still unknown. We tested whether CRH induced keratinocyte proliferation via interaction with CRH R1. Expression of CRH-R1 mRNA in the human keratinocyte and HSC-2, keratinocyte cell line, was analyzed by RT-PCR. The human keratinocyte and HSC-2 were recognized to have CRH-R1 expression ability. CRH signal is transduced into a cAMP-activated metabolic pathway via interaction with CRH-R1. Radioimmunoassay indicated that CRH binds to CRH receptor in HSC-2 cell when activating the metabolic pathway. Using thymidine incorporation assay, CRH had proliferative effect to HSC-2. This study suggests that CRH induces the proliferation of keratinocytes via interation with CRH receptors.     

Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).     

Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor

Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1alpha, 1beta, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2alpha (but not R2beta or R2gamma) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-alpha release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.     

Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes

Acne is a common skin disorder of the pilosebaceous unit. In addition to genetic, hormonal and environmental factors, abnormal colonization by Propionibacterium acnes has been implicated in the occurrence of acne via the induction of inflammatory mediators. To gain more insight into the role that sebocytes play in the innate immune response of the skin, particularly in acne, we compared the antimicrobial peptide and proinflammatory cytokine/chemokine expression at mRNA and protein levels, as well as the viability and differentiation of SZ95 sebocytes in response to co-culture with representative isolates of P. acnes type IA and type IB as well as Escherichia coli-derived lipopolysaccharide (LPS). We found that, in vitro, P. acnes type IA and IB isolates and LPS induced human beta-defensin-2 and proinflammatory cytokine/chemokine expression, and influenced sebocyte viability and differentiation. Our results provide evidence that sebocytes are capable of producing proinflammatory cytokines/chemokines and antimicrobial peptides, which may have a role in acne pathogenesis. Furthermore, since P. acnes types IA and IB differentially affect both the differentiation and viability of sebocytes, our data demonstrate that different strains of P. acnes vary in their capacity to stimulate an inflammatory response within the pilosebaceous follicle.     

Sebaceous epithelial cell differentiation requires cyclic adenosine monophosphate generation

The cyclic adenosine monophosphate (cAMP) generator choleratoxin is known to promote the growth of sebaceous epithelial cells (sebocytes) in monolayer culture in classical serum-containing media. Now that sebocytes can be grown in serum-free medium, we have examined whether choleratoxin or other cAMP generators are required for differentiation of rat preputial sebocytes in response to specific ligand activators of peroxisome proliferator-activated receptors (PPARs). Unexpectedly, choleratoxin reduced sebocyte proliferation. However, sebocyte differentiation in response to specific PPARalpha and PPARgamma agonists required a cAMP generator such as choleratoxin, and this response was suppressed by a protein kinase A inhibitor. In contrast, the stable prostacyclin analog, carbaprostacyclin (cPGI2), a PPARalpha,delta agonist that also generates cAMP, stimulated differentiation independently of choleratoxin. Furthermore, unlike the selective PPARalpha and PPARgamma agonists, cPGI2 stimulated both sebocyte DNA synthesis and proliferation. These data are compatible with the evidence that prostacyclin has the additional effect of generating cAMP. In addition, we addressed the possibility that choleratoxin may act as a surrogate for beta-adrenergic catecholamines in generating cAMP. In contrast with choleratoxin, both alpha- and beta-adrenergic catecholamines stimulated sebocyte growth and interfered with the choleratoxin effect on differentiation. These data suggest ligand-dependent, complex interactions between cAMP and the other signal transduction pathways involved in sebocyte growth and development.     

Peroxisome proliferator-activated receptors and skin development

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.     

Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades

Many important physiological roles of the urocortin (UCN) family of peptides as well as CRH involve the type 2 CRH receptor (CRH-R2) and downstream activation of multiple pathways. To characterize molecular determinants of CRH-R2 functional activity, we used HEK293 cells overexpressing recombinant CRH-R2beta and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2beta-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2beta activation induced transient beta-arrestin1 and beta-arrestin2, as well as clathrin, recruitment to the plasma membrane. Beta-arrestin2 appeared to be the main beta-arrestin subtype associated with the receptor. This was followed by CRH-R2beta endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2beta also induced transient activation of the ERK1/2 and p38MAPK signaling cascades that peaked at 5 min and returned to basal within 20-30 min. Unlike p38MAPK, activated ERK1/2 was localized both in the cytoplasm and nucleus. Experiments employing inhibitors of receptor endocytosis showed that CRH-R2beta-MAPK interaction does not require beta-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2beta C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2beta signaling because loss of a potential phospho-acceptor site in mutant receptors containing deletion or Ala substitution of the cassette TAAV resulted in reduced ERK1/2 activation and accelerated receptor internalization. These findings provide new insights about the signaling mechanisms regulating CRH-R2beta functional activity and determining its biological responses.     

Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways

We have previously demonstrated that corticotropin-releasing hormone (CRH) receptor 1 (CRH-R1) is functionally expressed in rat microglia. In the present study, we show that CRH, acting on CRH-R1, promoted cell proliferation and tumour necrosis factor-alpha (TNF-alpha) release in cultured rat microglia. Exogenous CRH resulted in an increase in BrdU incorporation compared with control cells, which was observed in a range of concentrations of CRH between 10 and 500 nm, with a maximal response at 50 nm. The effect of CRH on BrdU incorporation was inhibited by a CRH antagonist astressin but not by a cAMP-dependent protein kinase inhibitor H89. Exposure of microglial cells to CRH resulted in a transient and rapid increase in TNF-alpha release in a dose-dependent manner. In the presence of astressin, the effects of CRH on TNF-alpha release were attenuated. CRH effects on TNF-alpha release were also inhibited by specific inhibitors of MEK, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) (PD98059) or p38 mitogen-activated protein kinase (SB203580), but not by H89. Furthermore, CRH induced rapid phosphorylation of ERK and p38 kinases. Astressin, PD98059, and SB230580 were able to inhibit CRH-induced kinase phosphorylation. These results suggest that CRH induces cell proliferation and TNF-alpha release in cultured microglia via MAP kinase signalling pathways, thereby providing insight into the interactions between CRH and inflammatory mediators.