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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

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Journal of Cell Communication and Signaling Aims and scope

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.

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Abbreviations

α-SMA:

Alpha smooth muscle actin

CP:

Chronic pancreatitis

CCN2:

Connective tissue growth factor

ECM:

Extracellular matrix

GFP:

Green fluorescent protein

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HPRT1:

Hypoxanthine-guanine phosphoribosyl transferase 1

miR:

MicroRNA

PPiA:

Peptidylprolyl isomerase A

PSC:

Pancreatic stellate cell

qRT-PCR:

Quantitative real-time PCR

siRNA:

Small interfering RNA

TGFβ-1:

Transforming growth factor beta 1

TEM:

Transmission electron microscopy

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Acknowledgments

Supported by NIH grant R01 AA015554 awarded to D.R.B.

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Authors and Affiliations

  1. Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH, 43205, USA

    Alyssa Charrier, Ruju Chen, Li Chen, Sherri Kemper & David R. Brigstock

  2. Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, OH, 43210, USA

    Alyssa Charrier & David R. Brigstock

  3. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Takako Hattori & Masaharu Takigawa

  4. Department of Surgery, The Ohio State University, Columbus, OH, 43210, USA

    David R. Brigstock

Authors
  1. Alyssa Charrier
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  2. Ruju Chen
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  3. Li Chen
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  4. Sherri Kemper
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  5. Takako Hattori
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  6. Masaharu Takigawa
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  7. David R. Brigstock
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Correspondence to David R. Brigstock.

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Charrier, A., Chen, R., Chen, L. et al. 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. J. Cell Commun. Signal. 8, 147–156 (2014). https://doi.org/10.1007/s12079-014-0220-3

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  • DOI: https://doi.org/10.1007/s12079-014-0220-3

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