Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation

Abstract

Edited by Alex Toker Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ϳ20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active. This work was supported by the Norwegian Council for Cardiovascular Research (to O. J. K.) and Scientia fellowship program, University of Oslo (to A. K. G.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S7 and Tables S1 and S2.

Blocking CCN2 Reduces Progression of Sensorimotor Declines and Fibrosis in a Rat Model of Chronic Repetitive Overuse

“…NSAIDs may fail because they treat algesic symptoms but not the key underlying cause, which we and others believe are fibrotic adhesions tethering tissues and compressing nerves . The FG‐3019 antibody used here targets domain II of CCN2, the vWC domain, located within the amino‐terminal half of the full‐length protein . This domain is thought to stimulate collagen synthesis and bind proteoglycans in the extracellular matrix (other specific associations are being clarified) .…”

“…The FG‐3019 antibody used here targets domain II of CCN2, the vWC domain, located within the amino‐terminal half of the full‐length protein . This domain is thought to stimulate collagen synthesis and bind proteoglycans in the extracellular matrix (other specific associations are being clarified) . Systemic administration of FG‐3019 was used to block fibrogenic processes occurring in all upper extremity tissues involved in performing this chronic repetitive high force task, since collagen production increases in skeletal muscle, tendons, dermis, and extraneural connective tissues, and CCN2 increases in myofibers and small cells on their perimeter, as well as tenocytes, epitendon, mast, and Schwann cells .…”

“…Systemic administration of FG‐3019 was used to block fibrogenic processes occurring in all upper extremity tissues involved in performing this chronic repetitive high force task, since collagen production increases in skeletal muscle, tendons, dermis, and extraneural connective tissues, and CCN2 increases in myofibers and small cells on their perimeter, as well as tenocytes, epitendon, mast, and Schwann cells . The effects of blocking other domains of CCN2, such as domains III and IV shown to elicit cell signaling activity, may also reduce the observed multi‐tissue fibrosis, although this remains to be tested since to our knowledge no antibodies exist that block only specific domain functions.…”

“…Cellular communication network factor 2 (CCN2; CTGF) is a secreted matricellular protein with four modular domains that independently interact with different molecules, such as collagen and proteoglycans in the extracellular matrix . CCN1 (Cyr6) and CCN3 (NOV) are two family members that can have similar or opposing functions in fibrogenic processes as CCN2, respectively .…”

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury

“…CCN2 is a secreted matricellular protein with four modular domains that independently interact with different molecules, such as collagen and proteoglycans in the extracellular matrix 20 . CCN1/Cyr61 (also known as Cysteine‐rich angiogenic inducer 61) and CCN3/NOV (also known as nephroblastoma overexpressed) are two family members that can have similar or opposing functions in fibrogenic processes as CCN2, respectively 21‐23 .…”