AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair.

Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12-ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5' strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12-ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER.

Divergence of cAMP signalling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes.

Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signalling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signalling positions melanocytes to resist UV injury by upregulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia-mutated and Rad3-related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signalling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated upregulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (eg PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (eg MITF activation) are distinct.

Intrinsic muscle clock is necessary for musculoskeletal health.

KEY POINTS: The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1(-/-) ) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1(-/-) ), a model of advanced ageing. iMSBmal1(-/-) mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing. ABSTRACT: Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in ageing and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced ageing and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fibre levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle, is associated with a muscle phenotype including reductions in specific tension, increased oxidative fibre type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1(-/-) mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds the potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifying a novel crosstalk between skeletal muscle, cartilage and bone.

Long-term bowel dysfunction and decision regret in diverticulitis: A mixed methods study.

BACKGROUND: This study had aimed to describe long-term decision regret, bowel dysfunction, and the overall quality of life in patients with diverticulitis, and to determine if elective colectomy was associated with these patient-reported outcome measures. METHODS: This mixed-methods, survey-based study was administered to a national cohort of patients in the United States with diverticulitis. We measured decision regret (Brehaut Decision Regret), bowel dysfunction (Low Anterior Resection Syndrome score), and the overall quality of life (EuroQol 5 Dimension) in this population. We asked open-ended questions to elucidate factors that influenced patients' choices between elective colectomy and observation. RESULTS: Among the 614 respondents, 294 (48%) chose between colectomy and observational management, 94 (15%) had surgery, and 157 (26%) had major Low Anterior Resection Syndrome. Of the 294 that chose between colectomy and observational management, 51 (17%) experienced decision regret. Colectomy was associated with an average decrease in the Brehaut Decision Regret score by 6 points but was not associated with a categorical measure of decision regret (Brehaut Score ≥50). Bowel dysfunction and overall quality of life were not significantly associated with colectomy. Disease-related factors, psychosocial factors, and interactions with physicians were commonly cited as reasons for pursuing colectomy or observational management. CONCLUSION: Patients with self-reported diverticulitis describe high levels of decision regret and bowel dysfunction regardless of chosen management strategy. Physicians should be aware that psychosocial factors can strongly influence a patient's choice between colectomy and observational management. We advocated for future prospective studies using patient reported outcome metrics to improve outcomes in diverticulitis.

Melanocortin 1 Receptor: Structure, Function, and Regulation.

The melanocortin 1 receptor (MC1R) is a melanocytic Gs protein coupled receptor that regulates skin pigmentation, UV responses, and melanoma risk. It is a highly polymorphic gene, and loss of function correlates with a fair, UV-sensitive, and melanoma-prone phenotype due to defective epidermal melanization and sub-optimal DNA repair. MC1R signaling, achieved through adenylyl cyclase activation and generation of the second messenger cAMP, is hormonally controlled by the positive agonist melanocortin, the negative agonist agouti signaling protein, and the neutral antagonist ß-defensin 3. Activation of cAMP signaling up-regulates melanin production and deposition in the epidermis which functions to limit UV penetration into the skin and enhances nucleotide excision repair (NER), the genomic stability pathway responsible for clearing UV photolesions from DNA to avoid mutagenesis. Herein we review MC1R structure and function and summarize our laboratory's findings on the molecular mechanisms by which MC1R signaling impacts NER.

Defining the Contribution of MC1R Physiological Ligands to ATR Phosphorylation at Ser435, a Predictor of DNA Repair in Melanocytes.

The melanocortin 1 receptor (MC1R), a GS-coupled receptor that signals through cAMP and protein kinase A (PKA), regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions, and assess NER function. We report that melanocyte-stimulating hormone (α-MSH) or ACTH induce ATR-pS435, enhance XPA's association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human ß-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction, and reduce DNA repair. Although ASIP and HBD3 each blocked α-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

AIMS: Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production. RESULTS: We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown. INNOVATION: These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. CONCLUSION: Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.