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 nano-vesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected 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.

Fig. 1

MiR expression changes during experimental CP assessed by qRT-PCR. QRT-PCR for miR-148a, 802, 21, 199a-3p and 211* in RNA isolated from pancreata of mice treated with ethanol/cerulein or water/saline for 23 days. (* p < 0.01)

Fig. 2

Identification of miR-21 as being positively correlated with CCN2. Detection by qRT-PCR of a CCN2 mRNA or b miR-148a, 802, 21, 199a-3p or 211* in primary PSC treated on Day 5 of culture with 0–50 mM ethanol for 24 h. Detection by qRT-PCR of c CCN2 mRNA in PSC transfected with miR-802, 21, 199a-3p, 211* or miR-148 or d CCN2 mRNA after transfection of the cells with miR-21. e Venn diagram demonstrating the rationale for selection of miR-21 for subsequent studies

Fig. 3

Activated PSC are the principal producer of miR-21 transcript during experimental CP. Pancreatic sections from mice treated with water/saline a–b, water alone c–d, ethanol alone e–f or ethanol/cerulein g–h were evaluated by α-SMA immunohistochemistry and miR-21 in situ hybridization

Fig. 4

CCN2 drives miR-21 expression during autonomous PSC activation in vitro. a CCN2 and miR-21 expression during autonomous PSC activation in culture (Day 1–12) as demonstrated by qRT-PCR. b QRT-PCR for CCN2, collagen α1(I), or miR-21 in Day 8 PSC where CCN2 expression is inhibited by CCN2 siRNA

Fig. 5

CCN2 expression drives miR-21, which drives CCN2 expression in vitro. CCN2, miR-21 or collagen α1(I) expression levels as demonstrated by qRT-PCR in activated PSC (P5) transfected with CCN2 siRNA +/− pre-miR-21 for a 24 h or b 48 h. (*p < 0.05, **p < 0.001)

Fig. 6

MiR-21 knockdown inhibits CCN2 expression in PSC in vitro. QRT-PCR for miR-21 or CCN2 mRNA in PSC transfected with 0–100 nM miR-21 antagomir

Fig. 7

Characterization of PSC exosomes. Exosomes isolated from the medium of cultured PSC were analyzed by a Western blot for CD9 and b TEM. qRT-PCR was performed to detect miR-21 or CCN2 mRNA in c exosomes from activated PSC or d exosomes from PSC transfected for 24 h with either CCN2-GFP or miR-21-GFP. Values above the bars in d represent the approximate fold increase in transcript level versus their respective controls which are normalized to a value of 1 and had comparable ΔCT values to those shown in c

Fig. 8

Exosome-mediated uptake of miR-21 or CCN2 mRNA by PSC. Primary mouse PSC or rat SAM-K cells were incubated for 24 h with PKH26-stained exosomes isolated from their CCN2-GFP- or miR-21-GFP-transfected counterparts. The figure shows a phase contrast or fluorescence microscopy of recipient SAM-K cells or b qRT-PCR for GFP in recipient PSC or SAM-K cells