Abstract
The exocrine pancreatic acinar cell is unique for its rapid protein synthesis and packaging in zymogen granules (ZGs). However, while crucial to the pathogenesis of pancreatitis, the signaling involved in the transit of proteins via the Golgi is poorly understood in these cells. Noting the evidence of c-Src in regulating transit of cargo via the Golgi in other systems, we explored this in acinar cells. Stimulation of ZG formation with dexamethasone activated Src and increased the Golgi area in acinar cells. c-Src localized to the microsomes of acinar cells on immunofluorescence and subcellular fractionation. While other Src family members had no effect on the Golgi markers P115 and GM130, active c-Src increased the Golgi area these stained, extending them into the ER. Src inhibition reduced amylase staining outside the Golgi and increased it in a stack like Golgi morphology. In vivo pharmacologic inhibition or acinar specific genetic deletion of c-Src reduced ZG number and staining of amylase in ZGs along with increasing amylase retention in the microsomal fraction. Morphologically this was associated with smaller Golgi stacks, and dilation of the endoplasmic reticulum. Therefore the role c-Src regulated Golgi function, ZG formation and microsomal zymogen transit in acinar cells needs to be explored in pancreatitis.
Highlights
While pancreatic acinar cells have one of the most rapid protein synthetic and packaging machineries[1], the mechanisms regulating zymogen granule (ZG) formation- the organelles in which these proteins are stored for regulated secretion are not well understood
Staining of endogenous c-Src in mouse pancreatic tissue showed this to predominantly co-localize with the cis-Golgi marker GM 130 in exocrine acinar cells (Fig. 1A), though it extended both apically and basally around it. This was verified with another Golgi marker P115 in isolated acinar cells (Fig. 1B), and while the two did co-localize, c-Src showed a diffuse pan cytoplasmic appearance consistent with the endoplasmic reticulum (ER)
This localization was not noted for the Src family member Yes, which has been previously shown to be present in acinar cells
Summary
While pancreatic acinar cells have one of the most rapid protein synthetic and packaging machineries[1], the mechanisms regulating zymogen granule (ZG) formation- the organelles in which these proteins are stored for regulated secretion are not well understood. The cargo we studied is amylase, an abundant exocrine protein that is packed in zymogen granules and secreted in a polarized manner This was chosen to avoid the alternate trafficking of lysosomal hydrolases into lysosomes and retrograde transport of ER resident proteins with a KDEL sequence[10]. While several Src family kinases have been identified in acinar cells including c-Src[12], Yes[13], Lyn[14] and Fyn[15], which have been known to regulate the actin cytoskeleton[13,15], adherens junction[16], endocytosis[12], cytosolic calcium signaling[17] in addition to secretion, and trypsinogen activation[8]; the tools used to study these have heavily relied on pharmacologic inhibition which is not specific for individual Src family members. Our data show that c-Src is present on the Golgi and ER of acinar cells and its activity regulates transit of amylase along the secretory pathway
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