Molecular events surrounding secretory granule biogenesis in transgenic hormone producing liver cell lines

Abstract

Secretory granule biogenesis describes the events leading up to the budding of a nascent granule from the trans Golgi network. Literature surrounding secretory granule biogenesis is conflicting and has generated much debate. This thesis aims to address the important issues of this debate by utilizing the insulin-producing liver cell line HUH7-ins. This cell line has been shown to synthesize, store and secrete mature insulin in response to glucose via the possession of secretory granules. Using microarraytechnology the gene expression profile of HUH7-ins cells was compared with parental HUH7 cells, hoping to identify possible candidate genes contributing to secretory granule biogenesis. 164 genes were shown to be differentially expressed although no known granulogenic protein exhibited a change in expression. The data did suggest anervous system differentiation event and implicates myosin Vc in the regulated secretion of insulin. HUH7-ins cells express a number of granulogenic protein mRNAs and while chromogranin B (CgB) protein level remained constant upon insulin expression, a significant increase in the level of chromogranin A (CgA) was observed,though the significance of this increase in expression is unknown. The over-expression of CgA in a clone of HUH7-ins that did not possess the regulated secretory pathway was unable to rescue the regulated secretory pathway, suggesting that CgA expression alone is unable to form secretory granules in our model. To determine if the secretory granule biogenesis seen in HUH7-ins cells was specific to insulin, three prohormones of different neuroendocrine origin were over-expressed in HUH7 cells; amylin ( cell),pancreatic polypeptide (pancreatic islet) and proopiomelanocortin (pituitary). None of these prohormones were able to form structures in the cytoplasm that resembled secretory granules by immunofluorescent microscopy, nor did they induce the expression of CgA. No prohormone was detected in cell lysates or conditioned media, raising the possibility that these exogenous prohormone aggregates are trafficked to the lysosomal/endosomal system for degradation. This study provides significant information regarding the genome-wide expression changes induced upon secretory granule biogenesis in a liver cell line, describes the lack of effect of CgA in this eventand suggests that secretory granule biogenesis in this liver cell line is specific to insulin