Browsing by Author "Lawson, Kelly"
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Item Role of NPHS Gene in Mammalian Myogenesis and Rhabdomyosarcoma(2017-01-17) Lawson, Kelly; Avirneni, Usha; Galindo, ReneRhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, is a malignancy of muscle-lineage myoblasts that are blocked from differentiating into syncytial muscle. Treatments for high-risk RMS have not improved for three decades, underscoring the need to elucidate the molecular underpinnings of the disease and design new precision drug therapies. Using complementary model systems, the lab has uncovered misregulated myoblast cell-cell fusion signaling as a driver of RMS. As the machinery necessary for mammalian myoblast fusion is largely unknown, the lab has been probing for the myoblast cell membrane elements that facilitate RMS pathogenesis. We have generated preliminary data pointing towards Immunoglobulin Superfamily transmembrane Receptors (Ig-S-R) family members KIRREL and NPHS as putative fusion regulators, expression for which appears altered in RMS. To further probe for Ig-S-R activity in myoblast fusion and RMS, we considered the following: (1) If KIRREL and/or NPHS orchestrate myoblast fusion, we hypothesize that myoblasts, when switched to Differentiation Medium, should express either or both of the Ig-S-Rs; (2) When RMS cells are reprogrammed in culture to undergo differentiation and fusion, we predict that misexpression of these Ig-S-Rs are restored to comparably normal patterns. To address (1), we have used qRT-PCR to profile Kirrel and NPHS in differentiating wild-type myoblasts, which showed that Kirrel and NPHS are expressed and regulated during the window that fusion occurs. We are now utilizing shRNA against NPHS, as well as generating NPHS-overexpressing myoblast cell lines, to test whether NPHS is essential for myoblast differentiation and fusion, or can interfere with fusion when misregulated. To address (2), the lab has turned to All-trans retinoic acid (ATRA), which is known to reprogram the RMS RD cell line to undergo terminal differentiation. To determine whether Ig-S-R expression is altered in ATRA-induced reprogramming of RMS myoblasts, the lab used immunoblot analysis to show that expression of NPHS in ATRA-treated RD cells is properly restored. These results suggest that dysregulated NPHS expression associates with RMS. We are now testing genetically modified RMS cell lines to further profile how Ig-S-R misexpression facilitates RMS. In summary, our ongoing studies argue for Ig-S-R orthologs as RMS pathogenesis underpinnings, findings that will be presented and discussed at the Medical Student Research Forum.Item Role of NPHS Gene in Mammalian Myogenesis and Rhabdomyosarcoma(2019-03-27) Lawson, Kelly; Galindo, Rene; Castrillon, Diego H.; Le, Lu Q.BACKGROUND: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma comprised histologically of neoplastic skeletal muscle-lineage precursors that fail to terminally differentiate or fuse into syncytial muscle. The underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain largely unclear. The lab has shown misregulated myoblast fusion activity as a molecular underpinning of RMS pathogenesis. Data point to Immunoglobulin Superfamily transmembrane Receptors family members KIRREL and NPHS as fusion receptors. OBJECTIVE: We hypothesize that KIRREL and NPHS are essential for muscle development, with alterations underlying RMS pathogenesis. METHODS: Here, we look at pattern of expression of NPHS throughout myogenesis and its effects on differentiation and fusion. We use a C2C12 murine myoblast cell line to evaluate the role of NPHS in muscle development. Pattern of expression is evaluated through a qPCR analysis to quantify levels of NPHS RNA throughout the six-day mammalian myogenesis process. Loss-of- function studies are achieved by transfecting C2C12 cells with vectors containing sequences necessary to express shRNA directed against NPHS RNA, effectively knocking down the NPHS gene product in these cells. Gain-of-function studies are achieved by transfecting C2C12 cells with vectors containing the NPHS gene. The myoblasts with NPHS knockdown and overexpression are observed throughout the six-day myogenesis process to assess for alteration in differentiation, fusion, or both differentiation and fusion. To better visualize the results, crystal violet stains and immunofluorescent stains are used. RESULTS: Quantification of NPHS RNA illustrates the precisely regulated expression of NPHS during myogenesis. Specifically, upregulation of the gene is seen during key points in differentiation and fusion, with a dramatic increase in NPHS expression on days three and six of the six-day myogenesis process. NPHS loss-of-function studies show a decrease in myoblast differentiation and fusion. When viewed on day four, myoblasts with reduced expression of NPHS showed a significant decrease (p < 0.01) in differentiation and fusion when compared to controls. NPHS gain-of-function studies show enhanced differentiation and fusion. Day four and day six myoblasts with increased expression of NPHS had significantly increased levels of differentiation and fusion when compared to the control myoblasts (p < 0.01). CONCLUSION: These results lead us to conclude that NPHS is critical for proper muscle development, and potentiates RMS, making it a prospective treatment target. With this knowledge as a foundation, the pathways of myogenesis and the RMS pathogenesis will continue to be elucidated. This information will allow for novel targeted treatments of RMS that will not carry the morbidity and mortality of traditional treatments such as surgery, radiation, and chemotherapy.