Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the G-protein coupled receptor 56 (GPR56) gene was transiently upregulated during the early fusion of human myoblasts. Human mutations in GPR56 cause the disease bilateral frontoparietal polymicrogyria (BFPP), however the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56−/− myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, loss of GPR56 expression in muscle cells results in decreases or delays in the expression of MyoD, myogenin, and NFATc2. Our data suggest that these abnormalities result from decreased GPR56-mediated SRE and NFAT signaling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase (CK) compared to wildtype. In agreement with these findings, clinical data from 13 BFPP patients revealed mild serum CK increase in only 2 patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this GPCR during muscle development and that the motor delay observed in these patients is likely not due to primary muscle abnormalities.

The melanocortin 1 receptor (MC1R) is a highly polymorphic G protein-coupled receptor, which is known to modulate pigmentation and inflammation. In the current study, we investigated the pharmacological effects of select single-nucleotide polymorphisms (SNPs) (V60L, R163Q, and F196L). After transient expression of MC1Rs in human embryonic kidney 293 cells, basal and ligand-induced cAMP signaling and mitogen-activated protein kinase (MAPK) activation were assessed by using luciferase reporter gene assays and Western blot analysis, respectively. All receptor variants showed decreased basal cAMP activity. With the V60L and F196L variants, the decrease in constitutive activity was attributable, at least in part, to a reduction in surface expression. The F196L variant also displayed a significant reduction in potency for both the peptide agonist ␣-melanocyte-stimulating hormone (␣-MSH) and the small-molecule agonist 1-[1-(3-methyl-L-histidyl-O-methyl-Dtyrosyl)-4-phenyl-4-piperidinyl]-1-butanone (BMS-470539). In MAPK signaling assays, the F196L variant showed decreased phospho-extracellular signal-regulated kinase levels after stimulation with either ␣-MSH or BMS-470539. In contrast, the R163Q variant displayed a selective loss of ␣-MSH-induced MAPK activation; whereas responsiveness to the small-molecule agonist BMS-470539 was preserved. Further assessment of MC1R variants in A549 cells, an in vitro model of inflammation, revealed an enhanced inflammatory response resulting from expression of the F196L variant (versus the wild-type MC1R). This alteration in function was restored by treatment with BMS-470539. Overall, these studies illustrate novel signaling profiles linked to distinct MC1R SNPs. Furthermore, our investigations highlight the potential for small-molecule drugs to rescue the function of MC1R variants that show reduced basal and/or ␣-MSH stimulated activity.

Caucasians with prevalence estimates of 2 to 8 cases per The prevalence of homozygous hereditary hemochro-1,000 population. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Approximately 70% of affected persons matosis (HHC) is estimated at 1:250 in Caucasian adults.possess HLA-A3 alloantigen. 5,[22][23][24][25][26][27][28] Simon et al. hypothesize Little is known about ethnic subpopulations that might that the genetic mutation leading to iron overload originally be at increased risk for this disease. HLA data have sugoccurred in the Celtic peoples and that the HLA marker for gested a Celtic origin for HHC. Screening for HHC was HHC might constitute a genetic tracer of the migratory patoffered to all employees of the Massachusetts Polaroid tern of these peoples. 29 More recently, a non-HLA-linked Corporation. Participants with a transferrin saturation form of iron overload has been described in South African of ú55% or ú45% and an elevated serum ferritin concenblacks. 30 tration on two screenings were referred for liver biopsy.Most studies have dealt with populations from ethnically The diagnosis of HHC was based on histological criteria, homogeneous areas (Table 1). [9][10][11][12][13][14][15][16][17][18][19][20][21]31 Detailed comparisons of quantitative hepatic iron determination, hepatic iron inethnic and racial backgrounds have not been reported. The dex, and the phlebotomy requirement for iron depletion.current study examines the prevalence of HHC in a healthy Participants completed a questionnaire regarding their working population of heterogeneous ethnic and racial backethnic background. Two thousand two hundred ninetyground. We sought to identify ethnic and racial subpopulafour employees were screened, and 5 cases of HHC were tions that might be at high risk for inheriting this disease. tion of HHC with Celtic background (P Å .012). The esti-Polaroid study of prostate-specific antigen values in male employees mated cost of screening per patient identified was over age 50 years, and an additional 1,331 nonduplicate serum sam-$18,041. Polaroid Corporation has a high representation ples were collected through a corporation-wide informational and of employees of British-Irish ancestry. Our data suggest promotional campaign offering free screening for HHC. This included that they are at high risk for developing HHC. A signifi-a telephone hotline outlining the signs and symptoms of HHC and cant association of HHC with Celtic ancestry was found the benefits of early detection. Demographic data for the total Polarin this subpopulation, supporting the concept of a Celtic oid population were made available for comparison ( HLA-A and -B phenotyping of T-lymphocytes was performed on TS, transferrin saturation; HII, hepatic iron index; SF, serum ferritin.probands in anticipation of family screening. 43,44 From the 1 Division of Gastroenterology, Faulkner Hospital, Tufts University School of Medicine, Boston, MA; 2 Medical Department, Polaroid Corporation, Cambridge, MA;Protocol. The initial sc...

One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently test obesity therapeutics.