The CYR61/CTGF/NOV (CCN) family members induce endoplasmic reticulum stress and unfolded protein response

Background: The endoplasmic reticulum (ER) is the site of synthesis and folding of secreted membrane-bound and diverse organelle-targeted proteins. This process requires several factors, including ATP, Ca2+ and an oxidizing environment for proper disulphide-bond formation. The ER is sensitive to stress factors that perturb cellular energy levels, cellular redox state, or intracellular Ca2+ content. As a consequence, these alterations reduce the protein folding capacity of the ER leading to accumulation and aggregation of unfolded proteins ending an orchestrated response which is known as unfolded protein response (UPR). We and others have shown that matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover [1 – 3]. They share a similar architecture and contain a high content of cysteine residues. Previously, we have shown that large quantities of CCN1/CYR61 induce reactive oxygen species formation in portal myofibroblasts and ER stress in hepatic stellate cells (HSC) [1, 2].

Methods: We here used adenoviral-mediated gene transfer of CCN2/CTGF, CCN3/NOV and CCN4/WISP-1 in primary HSC and hepatocytes. Expression analysis of ER-stress related genes (Chop, Ire1α, Perk, Bip, Grp94, Dnajc3, Dnajb9, Trib) and splicing of Xbp-1 was done by qRT-PCR. Cellular senescence and apoptosis was measured by β-galactosidase and TUNEL assay. In vitro and in vivo expression of adenoviral constructs was done as described elsewhere [3].

Results: We found that similar to CCN1, the three other members of the CCN protein family induce splicing of Xbp-1 (L) to Xbp-1 (S) initiating ER stress and UPR when expressed in high quantities. In addition, typical marker genes of ER-stress and UPR (BIP, IRE1α, PDI, Chop) are induced in cells that express high quantities of CCNs. This capacity provides the basis for a physiological self-limiting process that restricts hepatic fibrogenesis. Moreover, intravenous injections of high quantities of Ad-CCNs via the tail vein were suitable to increase Bip and Chop mRNA expression and to decrease splicing of Xbp-1 (L).

Conclusions: The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. We suggest that the targeted transfer of adenoviral vectors triggering high expression of CCN proteins to profibrogenic cells is therapeutically effective to limit hepatic fibrosis.

References:

[1] Borkham-Kamphorst E, Schaffrath C, Van de Leur E, Haas U, Tihaa L, Meurer SK, Nevzorova YA, Liedtke C, Weiskirchen R. The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling. Biochim Biophys Acta 2014;1843:902 – 14.

[2] Borkham-Kamphorst E, Steffen BT, Van de Leur E, Haas U, Tihaa L, Friedman SL, Weiskirchen R. CCN1/CYR61 overexpression in hepatic stellate cells induces ER stress-related apoptosis. Cell Signal 2016;28:34 – 42.

[3] Borkham-Kamphorst E, Huss S, Van de Leur E, Haas U, Weiskirchen R. Adenoviral CCN3/NOV gene transfer fails to mitigate liver fibrosis in an experimental bile duct ligation model because of hepatocyte apoptosis. Liver Int. 2012;32:1342 – 53.