Modified Cutaneous Expression Of Melanocortin Receptor Type 2 (MC2R, ACTH Receptor) And Its Promoter SNPs In Patients With Alopecia Areata

Alopeica areata (AA) is a hair follicle-specific autoimmune disease with genetic predisposition and environmental trigger and AA is gradually accepted as an inflammatory skin disease responding to stress. Stress response is mediated by the stress system involved hypothalamic-pituitary-adrenal axis (HPA axis), sympathetic nervous system and immune system, in which HPA axis called stress axis plays a cardinal role. Growing studies showed that a decreased HPA axis response could cause increased susceptibility to autoimmune and inflammatory disease just as the glucocorticoid-deficient state.Except the classical central HPA axis, CRF and related peptides can act as local modulators of stress in the peripheral organs such as skin, gestational tissues, immune system, pancreas, liver, gastrointestinal tract, skeletal muscle, heart, lung and endocrine organs (ovaries, testes, adrenals, thyroid glands and ocular tissue. It has been grdually acknowledged that skin and hair follicle display peripheral HPA-axis-like signaling systems and the equivalent components of the HPA axis coordinate with neurotransmitters, neuropeptides and neurotrophins effectively modulate skin and immune cell functions such as cell proliferation, cytokine production and the hair cycle under normal and pathological conditions. Recently some studies showed that an insufficient HPA axis response involved in the pathogenesis of AA in spite that the pathology of stress-associated AA has not been well recognized.The HPA axis is organized in a hierarchical manner with feedback operating at several points along the axis. The elements of HPA axis titled stress hormones include corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH) and corticosteroids. Stress stimulates the HPA axis activity with an elevated secretion of CRH leading to increased ACTH secretion and corticosteroid production. These stress hormones binding with their cognate receptors activate the signaling hierarchy to respond stress. ACTH intermediates the HPA axis from CRH signal to cortisol secretion and is recognized to have a strongest effect on hair growth among all of the POMC peptides, so the ACTH specific recptor melanocortin receptor type 2 (MC2R or adrenocorticotropic hormone receptor, ACTHR) with its binding affinity restricted to ACTH may present a link between the physiological response to stressors and hair follicle-restricted immunological inflammation AA. To answer this critical question, we assessed AA lesions and normal human scalp tissue for the expression of MC2R.It is now generally accepted that AA fits the paradigm of a complex genetic trait, therefore genetic studies can be applied in study on AA pathogenesis. A genome-wide search revealed that a maximum multipoint LOD (logarithm of odds) score with most significant susceptibility position is in the region from 10cM to 30cM on chromosome 18. We searched genes of this region in GenBank and found the MC2R is just located in the range. Emerging evidences that single base mutation in the promoter region of MC2R gene can result in significantly reduced adrenal ACTH sensitivity and lowed glucocorticoid serum levels during ACTH infusion indicates that the polymorphism of the positions of the ACTH receptor gene may have a determining role in controlling glucocorticoid production in human. Furthermore several polymorphisms in promoter region of MC2R gene resulting in a lower ligand affinity with ACTH has also been reported in Chinese Han people. So our research is to investigate whether there was any association between MC2R promoter SNPs and stress-associated AA to decode the genetic mechanism of AA.Firstly we used Thomas Holmes and Richard Rahe's social readjustment rating scale to assess the association of psychosocial stress with AA patients, secondly MC2R protein was determined by immunohistochemistry in situ and western blotting quantificationally, thirdly MC2R mRNA level was detected with quantitative real-time RT-PCR, finally two SNPs were assessed by LDR at positions -759A>C and -2T>C in the MC2R promoter.The main results and conclusions were summarized as follows:1. 50.95% of patients with AA experienced at least three stressful events at the time and up to 6 months prior hair loss compared with 32.05% of controls within the six months before the enrolling (P<0.001). The SRRS results show patients with AA have a higher depression scores than controls (Z=6.628,P<0.01). The main stressful life events at risks for AA patients were changes of sleep, modification of habits, family troubles, problems of work and school as well as involvement of finance by logistic regression analysis. Our studies demonstrated that a significant elevation of psychological stress was felt by AA patients and there was a closely relationshiop between risk of AA and severe perception of some main stressful events in Chinese population.2. Immunohistochemistry demonstrated the MC2R protein was widely expressed on cell membrane and in cytoplasm in nearly all cutaneous compartments and was most prominently detectable in adnexal structures. Average MC2R protein levels in every section of skin were lower in AA lesions than in scalp tissue from normal controls and the most differences were showed in outer root sheath, hair bulb and the stratum spinosum and stratum granulosum of epidermis. The reduced expression of MC2R in the epithelium of AA lesions is remarkable. Thus we suggested that the modified expression of MC2R is almost comprehensively participated in the histopathology of AA and particularly involved in epithelial pathology of AA.3. Western blotting results demonstrated that the expression of total MC2R protein was decreased in lesions of AA than scalp tissues from healthy controls (P<0.05). The data confirmed the findings of immunohistochemistry quantificationally.4. There was low expression of MC2R mRNA in normal scalp tissue. About 5-fold increased MC2R mRNA expression in lesion from patients with AA was detected compared with that in scalp tissue from cortols (P<0.01).The findings 3, 4 demonstrated that there were robust differences in MC2R expression between healthy controls with AA patients. While mRNA levels were increased in lesions from AA patients compared with scalp tissues from normal controls, the protein levers of MC2R were decreased. We hypothesized that the contrary expression of MC2R on gene and protein lever just coincided with the evidence that an over-responsive HPA activity coexists with a deficient HPA response in AA. We proposed that the failed translation of MC2R mRNA to protein may be a crucial factor for the local insufficient HPA response in AA.5. Two SNPs were selected at position -759A>C and -2T>C in the MC2R promoter with accession numbers of rs1893220 and rs79533878. There was a significant difference between AA patients with stress and controls in the frequency of the MC2R rs79533878 (-2T>C) genotype CC+TC (41.1% vesus 27.8%,P=0.009, OR=1.252, 95%CI= 1.045～ 1.501). The novel MC2R promoter variants rs79533878 (-2T>C) significantly associated with the stress-associated AA (P=0.002, OR=1.576, 95%CI=1.148～2.162). However the allele frequencies at position -759A>C in the MC2R promoter did not show statistically differences between the patients and control groups. Our findings implicated that the polymorphism of the rs79533878 (-2T>C) in MC2R gene may be one important factor that are correlated with the modified HPA axis response to stress in AA patients in China.In conclusion: Our results demonstrated that AA in some patients was associated with stress. We suggested an over-responsiveness of HPA axis to stress might trigger the first episode of AA considering the reportedly elevated CRH, ACTH and the high MC2R mRNA expressin in lesion of AA, but the lower levels of MC2R protein, resulting in a deficit of ACTH/MC2R activity in skin could attenuate CRH singnal transmission and reduce HPA axis response locally in AA. We proposed the deficient translation of MC2R mRNA to protein may be a key factor for the local insufficient HPA response and shortage of cortisol. Our investigation also showed the novel MC2R promoter variant rs79533878 (-2T>C) significantly associates with some stress-associated AA and we suggested that the polymorphism of the MC2R rs79533878 (-2T>C) in promoter might be one critical factor that influences the altered HPA axis activity in AA. Our research would bring new knowledge into understanding of stress-associated hair-loss and would help design new approaches for the treatment of stress-associated AA in future.