Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca2+]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca2+]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca2+]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing

Computational solutions for omics data

High-throughput experimental technologies are generating increasingly massive and complex genomic data sets. The sheer enormity and heterogeneity of these data threaten to make the arising problems computationally infeasible. Fortunately, powerful algorithmic techniques lead to software that can answer important biomedical questions in practice. In this Review, we sample the algorithmic landscape, focusing on state-of-the-art techniques, the understanding of which will aid the bench biologist in analysing omics data. We spotlight specific examples that have facilitated and enriched analyses of sequence, transcriptomic and network data sets.National Institutes of Health (U.S.) (Grant GM081871

STS induces keratinocyte migration and <i>STC1</i> mRNA expression.

<p>(A) In the cell spreading assay, e-lam formation on fibronectin-coated plate was induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM) for 24 h. The cell images were captured under magnification of 200x (<i>top</i>). (B) Cell migration through the transwell was induced by 5 nM STS for 24 h and the cell images were captured under 100x magnification (<i>top</i>). (C) Western blot analysis demonstrated the increase of FAK phosphorylation (pY397) upon 5 nM STS treatment from 10–360 s. (D) Cell migration was increased upon STS treatment at 24 h, but was blocked by 2 µM PF573228. Cell images were captured under 100x magnification (<i>top</i>). (E) <i>STC1</i> mRNA and protein expressions were induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM). (F) STS-induced <i>STC1</i> mRNA expression was not significantly abolished by PF573228 at 24 h. Asterisks (***) denote <i>p</i><0.0001, (**) denote <i>p</i><0.005 and (*) denote <i>p</i><0.01 as compared to the respective control treatment.</p

Akt-GSK3β signaling mediates the keratinocyte migration process.

<p>(A) The inhibitory effects of 30 mM LiCl and 10 µM SH6 on GSK3β and Akt-signaling respectively, were determined by probing the phosphorylated and total forms of GSK3β and Akt. The band intensities were measured and the ratios of phosphorylated/total form were plotted. Bars with the same letter are not significantly different according to the results of one-way ANOVA followed by Duncan’s multiple range tests (<i>p</i><0.05). STS-induced (B) e-lam formation and (C) cell migration were suppressed by LiCl but were induced by SH6. The cell images were captured under 200x and 100x magnification for e-lam formation and cell migration, respectively (<i>top</i>). (D) STS-induced <i>STC1</i> mRNA expression was inhibited by LiCl, but was increased by SH6 (vs STS alone). Asterisks (*) denote <i>p</i><0.05 as compared to their corresponding control.</p

STC1 enhances STS-mediated re-epithelialization process.

<p>(A) After transient transfection of V5-tagged STC1/pLenti (STC1/pLenti) and empty vector control (pLenti) into the HaCaT for 24 h, the cells were treated with 5 nM STS for 24 h and the protein expression levels of V5, GSK3β and Akt were determined using western blotting. The band intensities were measured and the ratios of the respective phosphorylated/total proteins were plotted. STC1 overexpression was found to have synergistic effect on STS-inhibited phosphorylation of Akt. STS-induced (B) e-lam formation on fibronectin-coated plate and (C) cell migration were synergistically induced by the transient overexpression of STC1 (vs STS-treated pLenti). The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) (<i>top</i>). (D) In the scratched wound healing assay, the scratched wound was closed significantly more rapidly in the cells maintained in the conditioned medium (CM) containing overexpressed STC1 protein (CM-STC1) than the control medium (CM Ctrl). The cell images were captured under 100x magnification. The levels of STC1 proteins in the conditioned media (CM-Ctrl and CM-STC1) were determined from day1 to day3 using western blotting. (<i>top</i>); CM-Ctrl depicts as C and CM-STC1 depicts as T. Asterisks (**) denote <i>p</i><0.01 and (*) denote <i>p</i><0.05 as compared to STS-treated pLenti-transfected cells.</p

STC1 knockdown hinders STS-mediated re-epithelialization process.

<p>(A) After the transfection of STC1 siRNA for 48 h, the knockdown efficiency was examined by real-time PCR. In STC1 siRNA-transfected cells, STS-induced (B) cell migration and (C) e-lam formation on the fibronectin-coated plates were significantly reduced as compared to the NS siRNA-transfected cells. The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) (<i>top</i>). Under the STS+LiCl or STS+SH6 cotreatment, (D) the number of migrated cells and (E) e-lam formation on fibronectin-coated plate were compared between the STC1 siRNA-transfected cells and the NS siRNA-transfected cells. The knockdown of STC1 was found to inhibit cell migration induced by the STS + LiCl/SH6 cotreatments. The synergistic effect of STS/SH6 on the increase of e-lam formation was significantly reduced in the STC1 siRNA transfected cells. Asterisks (**) denote <i>p</i><0.01 and (*) denote <i>p</i><0.05 as compared to their corresponding NS siRNA-transfected cells.</p

STS-induced migration is mediated by intracellular Ca²⁺ influx.

<p>(A) STS treatment increased [Ca²⁺]i as measured using Ca²⁺ detection dye, Fura 2-AM. (B) The number of migrated cells was significantly reduced by 10 µM BAPTA/AM at 24 h (vs Ctrl) and the cell images were captured under 100x magnification (<i>top</i>). (C) STS-induced <i>STC1</i> mRNA was further induced by 1 µM ionomycin but was downregulated by 10 µM BAPTA/AM at 24 h of the cotreatments. Asterisks (*) denote <i>p</i><0.05 as compared to the Ctrl.</p

Early embryogenesis in zebrafish is affected by bisphenol A exposure

Summary Exposure of a developing embryo or fetus to endocrine disrupting chemicals (EDCs) has been hypothesized to increase the propensity of an individual to develop a disease or dysfunction in his/her later life. Although it is important to understand the effects of EDCs on early development in animals, sufficient information about these effects is not available thus far. This is probably because of the technical difficulties in tracing the continuous developmental changes at different stages of mammalian embryos. The zebrafish, an excellent model currently used in developmental biology, provides new insights to the field of toxicological studies. We used the standard whole-mount in situ hybridization screening protocol to determine the early developmental defects in zebrafish embryos exposed to the ubiquitous pollutant, bisphenol A (BPA). Three stages (60–75% epiboly, 8–10 somite, and prim-5) were selected for in situ screening of different molecular markers, whereas BPA exposure altered early dorsoventral (DV) patterning, segmentation, and brain development in zebrafish embryos within 24 hours of exposure

Stanniocalcin-1 Reduces Tumor Size in Human Hepatocellular Carcinoma

<div><p>Growing evidence has revealed high expression levels of stanniocalcin-1 (STC1) in different types of human cancers. Numerous experimental studies using cancer cell lines demonstrated the involvement of STC1 in inflammatory and apoptotic processes; however the role of STC1 in carcinogenesis remains elusive. Hepatocellular carcinoma (HCC) an exemplified model of inflammation-related cancer, represents a paradigm of studying the association between STC1 and tumor development. Therefore, we conducted a statistical analysis on the expression levels of STC1 using clinicopathological data from 216 HCC patients. We found that STC1 was upregulated in the tumor tissues and its expression levels was positively correlated with the levels of interleukin (IL)-6 and IL-8. Intriguingly tumors with greater expression levels of STC1 (tumor/normal ≥ 2) were significantly smaller than the lower level (tumor/normal<2) samples (p = 0.008). A pharmacological approach was implemented to reveal the functional correlation between STC1 and the ILs in the HCC cell-lines. IL-6 and IL-8 treatment of Hep3B cells induced STC1 expression. Lentiviral-based STC1 overexpression in Hep3B and MHCC-97L cells however showed inhibitory action on the pro-migratory effects of IL-6 and IL-8 and reduced size of tumor spheroids. The inhibitory effect of STC1 on tumor growth was confirmed <i>in vivo</i> using the stable STC1-overexpressing 97L cells on a mouse xenograft model. Genetic analysis of the xenografts derived from the STC1-overexpressing 97L cells, showed upregulation of the pro-apoptotic genes interleukin-12 and NOD-like receptor family, pyrin domain-containing 3. Collectively, the anti-inflammatory and pro-apoptotic functions of STC1 were suggested to relate its inhibitory effect on the growth of HCC cells. This study supports the notion that STC1 may be a potential therapeutic target for inflammatory tumors in HCC patients.</p></div

STC1 suppressed HCC tumor growth <i>in vitro</i>.

<p>Spheroid formation and cell migration assay were performed in both stably STC1-overexpressed Hep3B (A-B) and 97L cell lines (C-D). The sizes of spheroids were measured at 10 days of the incubation. At least 3 random fields representing 200 spheroids were counted under 200x magnification. (A) In Hep3B cells, the representative pictures of spheroids were shown (<i>top</i>). (C) In 97L cells, overexpression of STC1 was confirmed by probing with V5 antibody (<i>right</i>). Significant reductions in sizes of the spheroids in Hep3B/STC1 and 97L/STC1 were noted. * p<0.05, **p<0.01, compared with empty vector transduced cells. Bars = 50 μm. The activities of cell migration in transwells were induced in (B) Hep3B cells and (D) 97L cells by the treatment with 50ng/ml of IL6 or IL8 for 24h. At least 5 random fields were counted under 100x magnification. The pro-migratory effects of IL6 or IL8 on Hep3B/STC1 and 97L/STC1 were significantly suppressed by STC1-overexpression. * p<0.05, ** p<0.001, compared with the treatments in the respective pLenti-transduced cells; # p<0.00001, compared with the same treatment group.</p