Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

<div><p>Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—<u>h</u>eat <u>s</u>hock <u>p</u>rotein 90 <u>a</u>lpha (Hsp90α)—<u>l</u>ow density lipoprotein <u>r</u>eceptor-related <u>p</u>rotein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-<u>d</u>i<u>m</u>ethyl<u>a</u>minoethylamino-17demethoxy<u>g</u>eldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (<i>p</i><0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management.</p></div

HE staining showing the histological changes of mouse skin after deep second-degree burn injury.

<p>(<b>A</b>) Normal skin. (<b>B</b>) Burned skin from mice suffering from hot steam for 4 s. Deep partial-thickness burn injury was observed and hair follicles were not damaged. Yellow arrowed lines indicated the thickness of skin in unburned and burned mice. Red arrows indicated hair follicles.</p

IHC assay showing the changes of Hsp90α immunostaining in burned mouse skin.

<p>(<b>A</b>) Unwound normal skin showed a few positive Hsp90α stainings. (<b>C</b>, <b>E</b>) Epidermal and dermal tissues after burn injury appeared more positive brownish stainings, indicating that Hsp90α was induced after the burn stimulation. In addition, Hsp90α level was the highest at 12 h post-treatment (<b>C</b>) and somewhat decreased at 48 h (<b>E</b>). Magnification of red boxed areas in (<b>A</b>), (<b>C</b>) and (<b>E</b>) was shown as (<b>B</b>), (<b>D</b>)and (<b>F</b>), respectively.</p

An <i>in vitro</i> scratch assay showing the effects of Hsp90α on the migration of heat-shocked cells.

<p>Images were taken at the indicated time of incubation. Hsp90α-treated group showed more rapid reduction in the gap size at each time point tested than that in the control group, while 17-DMAG group showed slower gap closure even than the control (<i>p</i><0.05). AG, average gap, normalized to the gap size at 0 h.</p

Image_4_Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development.TIF

<p>SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J^−/− mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J^−/− macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J^−/− mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.</p

Anti-Fibrotic Actions of Interleukin-10 against Hypertrophic Scarring by Activation of PI3K/AKT and STAT3 Signaling Pathways in Scar-Forming Fibroblasts

<div><p>Background</p><p>The hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-β1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions.</p><p>Objective</p><p>To explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10.</p><p>Methods</p><p>Expression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins.</p><p>Results</p><p>HS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs.</p><p>Conclusion</p><p>IL-10 apparently inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.</p></div

Image_1_Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development.TIF

<p>SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J^−/− mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J^−/− macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J^−/− mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.</p

Image_3_Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development.TIF

<p>SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J^−/− mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J^−/− macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J^−/− mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.</p

IL-10 reduces fibrosis by activating PI3K and STAT3 pathways in HSFs.

<p>(a) Inhibition of IL-10-mediated attenuation of fibrotic markers in HSFs by IL-10RB (1∶500 dilution) at 48 h after the addition of IL-10 (20 ng/ml), IL-10RB, or both. After treatment, cells were harvested and analyzed by Western blotting. (b) Data are expressed as the mean ± SEM (<i>n</i> = 3, *<i>p</i><0.05 <i>vs.</i> control, ^#<i>p</i><0.05 <i>vs.</i> IL-10RB-treated HSFs). (c) Inhibition of IL-10-mediated attenuation of fibrotic markers in HSFs by LY294002 (50 µM) at 48 h after the addition of IL-10 (20 ng/ml), LY294002, or both. After treatment, cells were harvested and analyzed by Western blotting. (d) Data are expressed as the mean ± SEM (<i>n</i> = 3, *<i>p</i><0.05 <i>vs</i>. control, ^#<i>p</i><0.05 <i>vs</i>. LY294002-treated HSFs).</p

Immunohistochemical analysis of IL-10Rα expression in HS and NS tissue.

<p>Streptavidin-peroxidase DAB staining showed that IL-10Rα was localized in HS tissue (a–c) and NS tissue (d–f). IL-10Rα was distributed on the cell membrane and in the cytoplasm, with more intensive staining in NS (d–f) than in HS tissue (a–c). Scale bars, a and d, 100 µm; b and e, 50 µm; c and f, 25 µm.</p