NFkappaB activation is essential for miR-21 induction by TGFß1 in high glucose conditions.

Transforming growth factor beta1 (TGFß1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFß1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFß1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFß1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFß1 in high glucose conditions. The induction by TGFß1 was dependent on NFκB activation and subsequent ROS generation. TGFß1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFß1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFß1-NFκB-miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

Regulation of growth factors-associated cell migration by C-phycocyanin scaffold in dermal wound healing.

1. The present study examined the role of C-phycocyanin (C-pc) in relation to growth factors and cell migration during wound healing. 2. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse growth factors in wound tissue showed overexpression (up to 10-fold) of growth factors, such as Cxcl12, Fgf18, Lefty 1, Lefty 2, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf 22, Mdk, Igf 2, transforming growth factor (TGF)-α 1 and interleukin (IL)-1ß showed an upregulation of more than 30-fold than the control groups. TGF-ß subfamily cytokine growth factors, such as Bmp 2, 4 and 8b, and other growth factors, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp2 and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1ß expression. 4. C-pc had no direct effect on keratinocyte migration. However, keratinocytes that were co-cultured with fibroblasts showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on keratinocyte migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

NFkappaB-dependent regulation of urokinase plasminogen activator by proanthocyanidin-rich grape seed extract: effect on invasion by prostate cancer cells.

Tumor invasion and metastasis present major obstacles to successful control of androgen-independent prostate cancer. Cell migration is a fundamental aspect of cancer cell metastasis. Urokinase plasminogen activator (uPA) system is implicated in cell migration and cancer metastasis and has potential to be developed as therapeutic target. In recent years, efficacy of dietary nutrients in preventing and curing cancer has gained increasing attention. One such promising candidate is proanthocyanidin-rich grape seed extract (GSE). We investigated the efficacy of GSE in regulating uPA expression and cell migration using highly metastatic androgen-independent PC3 prostate cancer cells as a model. GSE down-regulated uPA as a function of concentration. Additional studies showed that GSE inhibited DNA-binding activity of the transcription factor nuclear factor kappa B (NFkappaB), which in turn decreased NFkappaB-dependent uPA transcription. Invasion assays revealed the inhibitory effect of GSE on PC3 cell migration. These in-vitro experiments demonstrate the therapeutic property of GSE as an antimetastatic agent by targeting uPA.

Iron-mediated stability of PAI-1 mRNA in adenocarcinoma cells-involvement of a mRNA-binding nuclear protein.

This study reports the stability of mRNA of type-1 plasminogen activator inhibitor (PAI-1), the major physiologic inhibitor of plasminogen activation, by deferoxamine-aided iron deprivation, in PC3 adenocarcinoma cells. ELISA and Northern analyses studies revealed dose-dependent increase in PAI-1 expression by deferoxamine-treated cells. Co-treatment with ferric citrate quenched the effect of deferoxamine, confirming the role of iron in PAI-1 regulation. DRB-based RNA chase experiments suggested that post-transcriptional mechanism was involved in PAI-1 regulation. De-novo protein synthesis was necessary for this regulation. Electrophoretic mobility shift assay revealed the presence of a nuclear protein, binding to the 3'-UTR of PAI-1 mRNA in an iron-mediated manner. This is the first report of iron-mediated mRNA-protein interaction in PAI-1, involved in mRNA stability.

Downregulation of urokinase-type and tissue-type plasminogen activators in a rabbit model of renal ischemia/reperfusion.

Urokinase-type and tissue-type plasminogen activators (uPA, tPA) are key enzymes for starting the plasminogen system, which plays important roles in various physiological and pathological conditions. In order to examine the gene regulation in rabbit pathophysiological models we attempted to clone full-length cDNAs encoding uPA and tPA from kidney extracts of rabbit (Oryctolagus cuniculus) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The cloned rabbit uPA and tPA cDNAs were 2,350 and 2,561 bp in length, respectively, and the basic molecular structures predicted from the cDNAs were well-conserved compared with human uPA and tPA. In a rabbit model of renal ischemia/reperfusion (I/R), the expression of uPA and tPA mRNAs was down-regulated and that of their physiological inhibitor, type 1 plasminogen activator inhibitor, mRNA was up-regulated in ischemic kidney compared to non-ischemic kidney. In addition, fibrinolytic activity in ischemic kidney was lower than that in non-ischemic kidney. It is suggested that repression of fibrinolysis in the kidneys in rabbit I/R may contribute to the progression of renal damage in the model.