IGF-I overexpression stimulates CCN5/WISP2 expression in pancreatic -cells, which promotes cell proliferation and survival against streptozotocin

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 834-867-Islet Biology
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-849
Subrata Chowdhury*1, Xiao Wang2, Bing Li1, Coimbatore B Srikant1 and Jun-Li Liu1
1McGill University Health Centre, Montreal, QC, Canada, 2Shanghai Jiao Tong University, Shanghai, China
In order to explore novel targets of IGF-I action within the pancreatic islets, we have recently performed a whole-genome DNA microarray analysis from IGF-I overexpressing islets and found ~100 genes specifically up- or down-regulated. Prominent among them CCN5 mRNA level was increased 3-fold; its protein level was increased 2-fold in the islets isolated from MT-IGF mice. Dual-labeled immunofluorescence revealed that CCN5 was present at low level in the β-cells of wild-type islets and was significantly induced in response to IGF-I overexpression. CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) proteins are involved in cell adhesion and extracellular matrix remodelling, skeletal development and chondrogenesis, angiogenesis and wound repair, proliferation and tumorigenesis (2). Among them, CCN5 is a secreted protein of 29 kDa, known to play positive or negative roles in cell proliferation (3), its expression in breast cancer cells can be induced by steroid hormones, serum, EGF, phorbol esters and IGF-I (4). Although its expression in pancreatic acinar and ductal cells has been reported, that in the islet cells has not been documented. The finding that IGF-I treatment of mouse islets increased CCN5 mRNA level significantly suggests it exerts this action directly at the level of the β-cells. 

To define the role of CCN5 in islet function, we overexpressed its cDNA in insulinoma MIN6 cells using pcDNA3.1 vector and confirmed by Western blot analysis that the protein level in stably transfected cell lines (MIN6-CCN5) increased 30-fold compared to cells transfected with the empty vector (MIN6-VC). Using MTT cell viability assay, we detected a 2-fold increase in the cell numbers of three independent lines of MIN6-CCN5 cells compared to MIN6-VC cells indicating that CCN5 promotes cell proliferation. We further detected 2-3-fold increases in the level of cyclin D1, in the rates of Akt phosphorylation at Ser-473 and Erk1/2 phosphorylation in MIN6-CCN5 vs. MIN6-VC cells. It is established that β-cell replication is associated with increased cyclin D1 and CDK4 levels (1); deficiency in CDK4 or cyclin D2 results in reduced β-cell mass and type 1 diabetes. Our results suggest that CCN5 stimulates β-cell replication, by activating Akt and Erk1/2 kinases and increasing the levels of cyclin D1. Finally, MIN6-CCN5 cells were found to be resistant to streptozotocin-induced cell death as evidenced by the reductions in the % apoptotic cells and caspase-3 cleavage after 24 h treatment. In summary, we have shown that CCN5 is normally expressed in islet β-cells and IGF-I directly stimulates its expression. CCN5 overexpression accelerates the proliferation of MIN6 cells, activates Akt and/or Erk1/2 kinase, and inhibits streptozotocin-induced apoptosis. These findings suggest that increased CCN5 expression contribute to IGF-I-stimulated islet cell growth and/or survival.

1. Cozar-Castellano I, Takane KK, Bottino R, Balamurugan AN, Stewart AF: Induction of b-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of CDK4 and cyclin D1. Diabetes 2004; 53:149-159.    2. Holbourn KP, Acharya KR, Perbal B: The CCN family of proteins: structure–function relationships. Trends Biochem Sci 2008; 33:461-473.    3. Russo JW, Castellot JJ: CCN5: biology and pathophysiology. J Cell Commun Signal 2010; 4:119-130.    4. Dhar K, Banerjee S, Dhar G, Sengupta K, Banerjee SK: IGF-I induces WISP-2/CCN5 via multiple molecular cross-talks and is essential for mitogenic switch by IGF-I axis in estrogen receptor–positive breast tumor cells. Cancer Res 2007; 67:1520-1526

Nothing to Disclose: SC, XW, BL, CBS, JLL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: This work was supported by Canadian Diabetes Association and the National Science and Engineering Research Council of Canada.