Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population

Melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α‐melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole‐exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in‐frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist‐induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild‐type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss‐of‐function (LOF) allele, while p.Val174del displayed a partial LOF attribute.     

BDNF promoted osteoblast migration and fracture healing by up‐regulating integrin β1 via TrkB‐mediated ERK1/2 and AKT signalling

Brain‐derived neurotrophic factor (BDNF) has been reported to participate in fracture healing, whereas the mechanism is still unclear. Since osteoblast migration is important for fracture healing, investigating effects of BDNF on osteoblasts migration may help to reveal its mechanism. Here, MC3T3‐E1 cells were used in vitro while closed femur fracture mice were applied in vivo. Cells migration was assessed with Transwell assay. The protein expression was analysed by immunoblotting. X‐ray and Micro‐CT were performed at different time after fracture. Our results showed that BDNF promoted MC3T3‐E1 cells migration, integrin β1 expression and ERK1/2 and AKT phosphorylation. K252a, a specific inhibitor for TrkB, suppressed BDNF‐induced migration, integrin β1 expression and activation of ERK1/2 and AKT. PD98059 (an ERK1/2 inhibitor) and LY294002 (an AKT inhibitor) both inhibited BDNF‐induced migration and integrin β1 expression while integrin β1 blocking antibody only suppressed cell migration. X‐ray and Micro‐CT analyses showed that the adenoviral carried integrin β1 shRNA group had slower fracture healing at 7 and 21 days, but not 35 days compared to the control group. Thus, we proposed that BDNF stimulated MC3T3‐E1 cells migration by up‐regulating integrin β1 via TrkB mediated ERK1/2 and AKT signalling, and this may help to enhance the fracture healing.     

Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells

Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.     

Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

Knockdown of CkrL by shRNA deteriorates hypoxia/reoxygenation‐induced H9C2 cardiomyocyte apoptosis and survival inhibition Via Bax and downregulation of P‐Erk1/2

Integrin β1 subunit and its downstream molecule integrin‐linked kinase and focal adhesion kinase have been confirmed to be essential to cell survival and inhibition of apoptosis and hypoxia/reoxygenation (H/R)‐induced injuries in cardiomyocytes. However, it is still unclear whether CrkL [v‐crk avian sarcoma virus CT‐10 oncogene homolog (Crk)‐like], which acts also as a component of the integrin pathway, could also affect H/R‐induced injuries in the cardiomyocytes. The rat‐derived H9C2 cardiomyocytes were infected with a CrkL small hairpin RNA interference recombinant lentivirus, which knockdowns the endogenous CrkL expression in the cardiomyocytes. Apoptosis, cell proliferation and survival were examined in the H9C2 cardiomyocytes treated with either H/R or not. Results showed that knockdown of CrkL could significantly increase apoptosis and inhibition of the cell proliferation and survival and deteriorate the previously mentioned injuries induced by H/R. In contrast, overexpression of human CrkL could relieve the exacerbation of the previously mentioned injuries induced by CrkL knockdown in the H9C2 cardiomyocytes via regulation of Bax and extracellular signal‐regulated kinase1/2 (p‐ERK1/2). In conclusion, these results confirmed that knockdown of CrkL could deteriorate H/R‐induced apoptosis and cell survival inhibition in rat‐derived H9C2 cardiomyocytes via Bax and downregulation of p‐ERK1/2. It implies that CrkL could mitigate H/R‐induced injuries in the cardiomyocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

PTEN-mediated ERK1/2 inhibition and paradoxical cellular proliferation following Pnck overexpression

Pregnancy upregulated non-ubiquitous calmodulin kinase (Pnck), a novel calmodulin kinase, is significantly overexpressed in breast and renal cancers. We present evidence that at high cell density, overexpression of Pnck in HEK 293 cells inhibits serum-induced extracellular signal-regulated kinase (ERK1/ERK2) activation. ERK1/2 inhibition is calcium-dependent and Pnck kinase activity is required for ERK1/2 inhibition, since expression of a kinase-dead (K44A) and a catalytic loop phosphorylation mutant (T171A) Pnck protein is unable to inhibit ERK 1/2 activity. Ras is constitutively active at high cell density, and Pnck does not alter Ras activation, suggesting that Pnck inhibition of ERK1/2 activity is independent of Ras activity. Pnck inhibition of serum-induced ERK1/2 activity is lost in cells in which phosphatase and tensin homolog (PTEN) is suppressed, suggesting that Pnck inhibition of ERK1/2 activity is mediated by PTEN. Overexpression of protein phosphatase-active but lipid phosphatase-dead PTEN protein inhibits ERK1/2 activity in control cells and enhances Pnck-mediated ERK1/2 inhibition, suggesting that Pnck increases availability of protein phosphatase active PTEN for ERK1/2 inhibition. Pnck is a stress-responsive kinase; however, serum-induced p38 MAP kinase activity is also downregulated by Pnck in a Pnck kinase- and PTEN-dependent manner, similar to ERK1/2 inhibition. Pnck overexpression increases proliferation, which is inhibited by PTEN knockdown, implying that PTEN acts as a paradoxical promoter of proliferation in ERK1/2 and p38 MAP kinase phosphorylation-inhibited, Pnck-overexpressing cells. Overall, these data reveal a novel function of Pnck in the regulation of ERK1/2 and p38 MAP kinase activity and cell proliferation, which is mediated by paradoxical PTEN functions. The possible biological implications of these data are discussed.     

Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti‐oxidant with anti‐proliferative effects on multiple cancers. However, its ability to modulate gene‐specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non‐canonical STAT pathways to impose the gene‐specific induction of G1‐arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1‐arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non‐canonical STAT pathways, each with a specific role in TA‐induced anti‐cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA‐binding activity of STAT1 and in turn enhanced expression of p21^Waf1/Cip1. However, TA binds to EGF‐R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL‐2 DNA‐binding activity. As a result, the expression and mitochondrial localization of BCl‐2 are declined. This altered expression and localization of mitochondrial anti‐pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF‐R/Jak2/STAT1/3 and P38/STAT1/p21^Waf1/Cip1 pathways and induce G1‐arrest and intrinsic apoptosis in breast carcinomas.     

GIT1 is a novel MEK1–ERK1/2 scaffold that localizes to focal adhesions

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G‐protein‐coupled receptor kinase‐interacting protein 1) has been shown to bind paxillin and regulate focal‐adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen‐activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled‐coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co‐localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.     

In vitro behavior and UV response of melanocytes derived from carriers of CDKN2A mutations and MC1R variants

Coinheritance of germline mutation in cyclin‐dependent kinase inhibitor 2A (CDKN2A) and loss‐of‐function (LOF) melanocortin 1 receptor (MC1R) variants is clinically associated with exaggerated risk for melanoma. To understand the combined impact of these mutations, we established and tested primary human melanocyte cultures from different CDKN2A mutation carriers, expressing either wild‐type MC1R or MC1RLOF variant(s). These cultures expressed the CDKN2A product p16 (INK4A) and functional MC1R. Except for 32ins24 mutant melanocytes, the remaining cultures showed no detectable aberrations in proliferation or capacity for replicative senescence. Additionally, the latter cultures responded normally to ultraviolet radiation (UV) by cell cycle arrest, JNK, p38, and p53 activation, hydrogen peroxide generation, and repair of DNA photoproducts. We propose that malignant transformation of melanocytes expressing CDKN2A mutation and MC1RLOF allele(s) requires acquisition of somatic mutations facilitated by MC1R genotype or aberrant microenvironment due to CDKN2A mutation in keratinocytes and fibroblasts.     

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation

Activation of the melanocortin 1 receptor (MC1R) by α‐melanocortin (α‐MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)‐induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α‐MSH. α‐MSH up‐regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV‐induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3‐related (ATR) and ataxia telangiectasia mutated (ATM) and their respect‐ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild‐type p53‐induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α‐MSH increases UV‐induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.     

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

Long noncoding RNA HNF1A‐AS1 regulates proliferation and apoptosis of glioma through activation of the JNK signaling pathway via miR‐363‐3p/MAP2K4

Long noncoding RNAs (lncRNAs) have been proven to exert important functions in the various biological processes of human cancers. It has been reported that lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A‐AS1) was abnormally expressed and played a role in the initiation and development of various human cancers. In this study, we confirmed that the expression level of HNF1A‐AS1 was increased in glioma tissues and cells. Knockdown of HNF1A‐AS1 inhibited cell proliferation and promoted cell apoptosis in glioma. Then, we disclosed the downregulation of miR‐363‐3p in glioma tissues and cell lines. The interaction between HNF1A‐AS1 and miR‐363‐3p was identified in glioma cells. Furthermore, an inverse correlation between HNF1A‐AS1 and miR‐363‐3p was observed in glioma tissues. Afterwards, we recognized that MAP2K4 was a direct target of miR‐363‐3p. The expression of MAP2K4 was negatively correlated with miR‐363‐3p while positively related to HNF1A‐AS1 in glioma tissues. We also found the regulatory effect of HNF1A‐AS1 on the MAP2K4‐dependent JNK signaling pathway. All findings indicated that HNF1A‐AS1 induces the upregulation of MAP2K4 to activate the JNK signaling pathway to promote glioma cell growth by acting as a miR‐363‐3p sponge.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.