A66. Collagen Iα2 gene expression is regulated by the bHLH transcription factor scleraxis

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ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862 – 918

tidylinositol-3 kinase (PI3K) plays an important role in IPC protection. Our study examined the isoform specificity of PI3K in IPC using PI3Kg knockout (PI3Kg / ) and PI3Ka dominant-negative (PI3KaDN) mice. Left ventricular developed pressure (LVDP) was measured in isolated perfused hearts using the Langendorff apparatus. Recovery of myocardial function was assessed during 30 min of sustained global ischemia followed by 40 min of reperfusion (I-R) with or without 4 cycles of 5 min of ischemia and 5 min of reperfusion. Recovery rate of LVDP in IPC group was significantly higher (85.1 T 9.0%, n = 15) than I-R group in wild-type hearts (29.3 T1.9%, n = 11) (P < 0.05). However, the protective effect of IPC on LVDP was completely abolished in PI3Kg– /– hearts (36.9 T 4.1%, n = 12). Surprisingly, PI3KaDN hearts showed significant recovery after both I-R (90.1 T 7.7%, n = 15) and IPC (88.4 T 7.7%, n = 11) ( P < 0.05). Western blot analyses showed a significant reduction of PKB/Akt and GSK3h phosphorylation in wild-type hearts following I-R, but similar levels of protein phosphorylation in the IPC group compared to control normoxic hearts. Phosphorylation of these proteins was not reduced in either I-R or IPC PI3KaDN hearts compared to normoxic hearts, while significantly lower phosphorylated states of these proteins were observed PI3Kg / hearts. The protection in PI3KaDN hearts against ischemia was blocked by infusion of the PI3K inhibitor, wortmannin, (28.4 T 4.2%, n = 5). Our studies demonstrate PI3Kg-PKB/Akt-GSK3h signaling is important in IPC and for cell survival during I-R. doi:10.1016/j.yjmcc.2006.03.301

A94. Mechanisms of tnf induced IGF-1 production by human bone marrow stem cells Paul Crisostomo, Meijing Wang, Christine Herring, Daniel R. Meldrum. Indiana Univ. School of Medicine, Indianapolis, IN 46202 Objective: The plasticity of progenitor cells has resulted in positive remodeling and the regeneration of viable tissues. However, recent studies indicate that transplanted adult progenitor cells may not differentiate into recipient tissue. Progenitor cells may instead mediate their beneficial effects, limit apoptosis, and modulate inflammation via production of local factors. We hypothesize that tumor necrosis factor alpha (TNF): 1) activates human mesenchymal stem cells (hMSCs) to release protective growth factors; and 2) activates hMSCs by a p38 MAPK dependent mechanism. Methods: hMSCs (Cambrex Bio Science) were harvested and cultured from normal human bone marrow. After three passages, hMSCs were divided into 4 experimental groups (1  105 cells/well) and stressed: 1) without intervention; 2) with TNF (50 ng/ml); 3) with TNF and p38 MAPK inhibitor (SB203580, 10 AM); and 4) p38 MAPK inhibitor. After 24 h incubation, hMSC activation was determined by measuring supernatants for insulin like growth factor 1 (IGF-1) production by ELISA. The experiment was per-

formed on three separate occasions (n = 6 –11 wells/group). Differences considered significant if P < 0.05 by T-test. Results: TNF exposure increased hMSC IGF-1 production (143 T 38 pg/mg) in comparison to controls (92 T 16 pg/mg). Interestingly, TNF and p38 MAPK inhibitor, significantly (86%) increased IGF-1 expression (207 T 28 pg/mg) over TNF alone. Moreover, p38 MAPK inhibitor alone (182.1 T 29 pg/mg) significantly (106%) increased IGF-1 expression over controls. Conclusion: hMSCs release growth factors in response to both TNF and p38 MAPK inhibitor. Further understanding of the mechanisms of stem cell activation may result in the engineering of ‘‘super’’ stem cells to maximally repair damage tissue. doi:10.1016/j.yjmcc.2006.03.302

A66. Collagen IA2 gene expression is regulated by the bhlh transcription factor scleraxis L. Espira, L. Lamoureux, S.C. Jones, I.M.C. Dixon, M.P. Czubryt. Department of Physiology, University of Manitoba, Winnipeg, Manitoba Fibrosis is a pathological process in which cardiac fibroblasts and myofibroblasts become activated and begin the deposition of extracellular matrix components (e.g. collagens) following an infarct or other insult. Fibrosis has a detrimental effect on cardiac function by interfering with compliance and contractility. In a mouse model of acute cardiac failure, we observed the induction of scleraxis prior to the up-regulation of collagen genes. Scleraxis, a bHLH transcription factor, is expressed in the sclerotome during mouse embryonic development, where it is involved in chondrogenesis and skeletogenesis. We hypothesized that scleraxis regulates the expression of collagen genes, since it is normally expressed in areas of connective tissue development. To investigate this, COS7 cells were transfected with the collagen 1a2 gene promoter fused to a luciferase transgene with or without scleraxis, E12, E47 and Id2 expression vectors. Reporter assays showed that scleraxis strongly up-regulated the collagen Ia2 promoter. E12 and E47 failed to augment Scleraxis transactivation. Id2, an HLH protein which inhibits bHLH transcription factors, attenuated scleraxis transactivation of the collagen Ia2 promoter in a dose dependent manner. Transfection with MyoD, a related bHLH transcription factor, had no effect on promoter expression. Treatment of cardiac fibroblasts with TGF-h1, an inducer of fibrosis, resulted in upregulation of scleraxis expression. Our data shows that scleraxis directly up-regulates the collagen Ia2 promoter. Endogenous E proteins may co-regulate scleraxis, since scleraxis was inhibited by Id2. The inability of MyoD to up-regulate the reporter indicates that Scleraxis is a specific regulator of this promoter. Taken together these results show a link between scleraxis and collagen production by cardiac fibroblasts. This is the first time that a link has been demonstrated between scleraxis and cardiac fibrosis. Supported by CIHR. doi:10.1016/j.yjmcc.2006.03.303