Identification of ITGBL1: A Novel Regulator of Adipogenesis




Spurgin, Stephen B.

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BACKGROUND: Obesity is a global epidemic that increases the risk of chronic metabolic disease. The rising burden of human suffering inflicted by the double-edged sword of obesity and diabetes has increased the urgency of understanding all aspects of adipose tissue biology, including how adipocytes are formed from their progenitor cells (the preadipocyte) when the body has a need to store excess lipid. It has been shown that the pathological expansion of white adipose tissue (WAT) leads to insulin resistance and cardiovascular disease. In spite of this clear observation, the mechanisms driving the formation of new adipocytes in obesity remain unclear. The work done up to this point has shown that the presumptive preadipocytes resemble pericytes: mesenchymal cells embedded in the vascular basement membrane (1). The pericyte is defined by this anatomic location as well as by the expression of the cell-surface marker platelet-derived growth factor receptor β (PDGFRβ) (2). Recently, Gupta et al. have discovered that Zfp423, a key transcriptional regulator of preadipocyte determination, is expressed in a subset of adipose pericytes in the microvasculature of adult adipose tissues (3). Functional analyses of these cells have revealed that Zfp423+ pericytes serve as a subpopulation of committed preadipocytes in the adipocyte lineage. The ability to isolate committed preadipocytes in vivo and study them in vitro is an excellent tool for investigation and discovery of factors that drive adipocyte commitment and differentiation. OBJECTIVE: This research should identify new genes involved in the commitment or differentiation of adipocyte precursors. METHODS: The identification of Zfp423, a transcription factor, as a regulator of preadipocyte determination was a key step forward in understanding adipogenesis (4). Zfp423 regulates the expression of Pparγ, the "master regulator" of adipogenesis. To understand the localization of Zfp423 in vivo, Gupta et al. derived Zfp423GFP BAC transgenic reporter mice, which express GFP under the control of the Zfp423 locus. Interestingly, Zfp423 expression in adipose tissue is found in mature adipocytes and a subset of perivascular cells expressing PDGFRbeta, supporting a long-standing hypothesis of a perivascular location for preadipocytes (3). Having identified a committed preadipocyte in vivo using the Zfp423-GFP mouse, we used a microarray to compare gene expression between the population of non-committed mesenchymal stem cells (PDGFRbeta+; Zfp423-low) and committed adipocyte progenitor cells (PDGFRbeta+; Zfp423-high) in the mouse model. The top differentially expressed genes were identified and their expression was manipulated in preadipocytes in vitro to assess the effect of those genes on the ability of cells to become adipocytes. The most differentially regulated protein was ITGBL1, which was highly expressed in the Zfp423+ mural cells. Given its high expression in these primed early preadipocytes, we investigated the role of ITGBL1 in preadipocyte differentiation in vitro. Here, we show that shRNA or CRISPR-mediated inactivation of ITGBL1 expression increases the propensity of mesenchymal stem cells to undergo adipocyte differentiation. RESULTS: In order to properly assess the potential adipogenic role of the top regulated genes from the microarray analysis, I cloned the genes (see Table 3.1) from cDNA generated from the stromal-vascular fraction of the mouse inguinal fat pad. I then overexpressed the genes in both the 3T3-L1 preadipocyte cell line and the C3H 10T1/2 mesenchymal stem cell line using a retroviral vector (pMSCV-puro), and assessed whether the cells were then more or less likely to differentiate into mature adipocytes when treated with an adipogenic hormonal cocktail. The overexpression did not result in any clear, repeatable phenotypic changes. However, an investigation of knockdown of these genes did yield significant results. I cloned three shRNAs to each gene, as well as three CRISPR gRNAs, and examined the results of mRNA knockdown and genetic deletion of the genes. One gene, ITGBL1, stood out clearly from those I investigated by way of its robust and dramatic impact on adipogenesis. ITGBL1, or "Integrin beta-like protein 1," has not been previously described in any role with regard to the differentiation of mesenchymal stem cells. However, when the C3H 10T1/2 mesenchymal stem cell line was treated with either shITGBL1 or CRISPR-ITGBL1, there was a highly significant increase in adipogenesis in vitro. CONCLUSION: This work has shown that Itgbl1, a previously uncharacterized protein, is a novel repressor of adipogenesis. Through shRNA knockdown of gene expression and CRISPR-cas9 knockout of the gene, I have shown the role of Itgbl1 in preventing the differentiation of an established mesenchymal stem cell line into a mature fat cell in vitro. Further work will focus on the identification of additional genetic factors involved in adipogenesis using murine tools described in this thesis. Additionally, a human model of adipogenesis--the infantile hemangioma--will be utilized to show the relevance of these genes in the human model of disease.

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