Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes

Abstract

Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol-excessive consumption of which is a major cause of pancreatitis in the West-normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen α1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50-150 nm CD9-positive nanovesicles. Exosomes from CCN2-GFP-or miR-21-GFPtransfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.

Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p

“…11,19,60,61 Whereas most previous studies have focused on these mechanisms as they relate to exosomal pathways in cancer and immune cells, including HCCs, 15,17,18 emerging evidence from this and other laboratories has revealed that fibrogenic cells are also regulated by exosomal networks. 10,22,23,31,34 In addition to revealing that miR-199a-5p targets the CCN2 3 0 -UTR in the same cells as which it is produced, the studies reported here indicate that miR199a-5p is released from HSCs in exosomes and that exosomal miR-199a-5p concentrations reflect those of their producer cells, being expressed at high levels in exosomes from quiescent HSCs and at low levels in exosomes from activated HSCs. We found that delivery of endogenous exosomal miR-199a-5p from quiescent HSCs to activated HSCs resulted in down-regulation of CCN2 3 0 -UTR activity and that exosomes from quiescent HSCs are preferentially targeted to the activated HSC population in vivo in CCl 4 -injured liver and suppress fibrogenic gene expression in activated HSCs in vitro in a miR-199a-5pedependent manner.…”

“…We propose that exosomal dampening mechanisms help to maintain HSC quiescence and that exosomes from populations of quiescent HSCs contribute to suppression of HSC activation during localized or acute liver injury, during diminished periods of fibrogenic activity in progressive liver diseases, such as nonalcoholic steatohepatitis, and/or during therapy-induced fibrosis regression. Because exosomes from activated HSCs can deliver CCN2 mRNA to quiescent HSCs, thus increasing their fibrogenic potential, 34 it is likely that differentially activated HSCs engage in a functional exosome exchange, which allows their phenotypic status to be locally communicated and for the recipient cells to respond accordingly. Because it is now becoming recognized that HSC function can be stimulated by exosomes from other cell types, such as injured hepatocytes, 31 complex exosome communication networks likely exist in the liver that serve to choreograph and fine-tune the response to injury and maintain homeostasis under normal healthy conditions.…”

“…Our previous studies found that, when isolated from normal (nonfibrotic) animals, cells studied within 24 hours of brief culture do not exhibit characteristics typical of activated cells (eg, a-SMA, CCN2) and thereafter gradually transition to a highly activated phenotype during the ensuing 7 to 20 days of culture. 10,22,23,33,34 In this study, CCN2 and miR-199a-5p expression were measured in primary HSCs until D20 of culture. In some experiments, triplicate wells of cells in serum-free medium were incubated for up to 48 hours in the presence of 0 to 3 ng/mL of transforming growth factor (TGF)-b1.…”

“…Because we have reported previously that exosomes from activated or quiescent HSCs are involved in intercellular shuttling of, respectively, CCN2 34 or miR-214 and Twist1, 10,23 it was of interest to determine whether miR199a-5p is also an exosomal constituent. We have previously reported that HSC-derived exosomes are bimembrane vesicles (assessed by transmission electron microscopy) that have a charge of À26 mV and a mean diameter of approximately 140 nm (assessed by zeta potential analysis and dynamic light scattering, respectively) and are positive for the exosome markers CD9 and flotillin (assessed by Western blot).…”

Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217

“…Vasudevan et al (38) showed that when cells are serum starved, binding of miR-369-3 to a reporter mRNA (containing the TNF-a 39UTR) stimulates translation. In pancreatitis, increased expression of miR-21 was found to be associated with connective tissue growth factor (CCN2) upregulation (39). Overexpression of miR-542-3p in U2OS cells enhanced p53 expression and stimulated the expression of p53 targets (40).…”