CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2−/− chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2−/− chondrocytes, confirming a defect in ECM production. Ccn2−/− chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of α5 integrin. Moreover, CCN2 can bind to integrin α5β1 in chondrocytes and can stimulate increased expression of integrin α5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2−/− chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin α5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways

The 5′ untranslated regions (UTRs) of CCN1, CCN2, and CCN4 exhibit cryptic promoter activity

CCNs are structurally related matricellular proteins that are highly expressed in many embryonic and adult tissues, including the skeletal system and tumors, where canonical cap-dependent translation is suppressed under hypoxic environments. CCNs are encoded by mRNAs containing long G/C rich 5′-untranslated regions (5′-UTRs). Given that they are expressed under conditions of cellular stress, it has been suggested that the long G/C-rich regions contain internal ribosomal entry sites (IRES) that allow these mRNAS to be translated under conditions where cap-dependent translation is suppressed. Previously published work supported this possibility. However, recent studies have shown that a number of previously reported cellular IRES elements do not in fact possess IRES activity. Here we aimed to reveal whether the 5′UTRs of CCNs harbor IRES activities. The 5′UTRs of CCN1, 2, and 4 were tested in this study. Our results showed that the 5′UTRs of these genes do not contain IRES elements, but instead appear to contain cryptic promoters. Both promoterless and hairpin-containing dicistronic tests showed that transcription was initiated by cryptic promoter elements in 5′UTRs of CCN1, 2, and 4. When dicistronic mRNAs were translated in vitro or in vivo, no IRES activities were detected in the 5′UTRs of CCN1, 2, and 4. Furthermore, these cryptic promoter activities from 5′UTRs of CCN1, 2, and 4 could be detected in various cell types, including chondrocytes, osteoblasts, and endothelial cells, where the cryptic promoter permitted varying degrees of activation. In addition, the core promoter element of the CCN2 5′UTR was identified. CCNs are expressed under conditions of cellular stress, and it has been suggested that some CCN family members utilize IRES-mediated translation initiation to facilitate this expression. We found no evidence for IRES activity, but rather found that the unusually long 5′UTRs of CCNs 1, 2, and 4 harbor cryptic promoters that showed varying degrees of activity in different cell types. These results suggest that these promoters may contribute to the regulation of CCN genes in vivo