Adiponectin Regulates Cutaneous Wound Healing by Promoting Keratinocyte Proliferation and Migration via the ERK Signaling Pathway

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Adiponectin plays a pivotal role in the pathogenesis of diabetes and is considered to be involved in various pathological conditions associated with diabetes; however, its role in wound repair is unknown. In this study, we elucidated the involvement of adiponectin in cutaneous wound healing in vitro and in vivo. Normal human keratinocytes expressed adiponectin receptors, and adiponectin enhanced proliferation and migration of keratinocytes in vitro. This proliferative and migratory effect of adiponectin was mediated via AdipoR1/AdipoR2 and the ERK signaling pathway. Consistent with in vitro results, wound closure was significantly delayed in adiponectin-deficient mice compared with wild-type mice, and more importantly, keratinocyte proliferation and migration during wound repair were also impaired in adiponectin-deficient mice. Furthermore, both systemic and topical administration of adiponectin ameliorated impaired wound healing in adiponectin-deficient and diabetic db/db mice, respectively. Collectively, these results indicate that adiponectin is a potent mediator in the regulation of cutaneous wound healing. We propose that upregulation of systemic and/or local adiponectin levels is a potential and very promising therapeutic approach for dealing with diabetic wounds.     

阻断Notch信号的角质形成细胞对成纤维细胞胶原合成的影响

目的:本课题组前期工作已经证明阻断Notch信号对角质形成细胞的分泌功能有所影响,使其分泌的多种促纤维化因子发生改变.本实验将探讨在复合培养的条件下,通过调节角质形成细胞中的Notch信号,了解其对成纤维细胞collagen-1合成的影响.方法:运用角质形成细胞-成纤维细胞复合培养的模型,于复合培养前3天进行血清刺激或者γ-分泌酶抑制剂DAPT阻断角质形成细胞中的Notch信号,即在角质形成细胞分化前进行干预.然后提升至气液交界面使其达到复层生长和终末分化,后与成纤维细胞共培养,观察阻断Notch信号后的角质形成细胞对成纤维细胞合成collagen-1的影响.结果:在分化前,给予角质形成细胞血清刺激,在复合培养0小时,Notch-1、Jagged-1表达明显升高(P＜0.05),在复合培养第一天,p21表达上调、p63表达下降(P＜0.05),表明在复合培养时,角质形成细胞中的Notch信号明显活化.通过在血清刺激前给予DAPT预处理,在复合培养0小时,角质形成细胞中Notch信号下游分子p21和p63的表达恢复至无血清刺激水平(P＜0.05),表明DAPT组确实阻断了角质形成细胞中的Notch信号.而DAPT组的成纤维细胞collagen-1的表达相对于血清刺激组明显下降,而与无血清组无明显差异,表明阻断Notch信号后的角质形成细胞在复合培养条件下,确实能够抑制成纤维细胞collagen-1的表达,使其合成collagen-1的量恢复至无血清刺激水平.结论:DAPT能够阻断Notch信号的活化,用阻断Notch信号后的角质形成细胞与成纤维细胞共培养,能够明显抑制成纤维细胞合成Ⅰ型胶原的能力,从而抑制瘢痕的增生.     

Laminin-5 Inhibits Human Keratinocyte Migration

Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the α3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the α3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.     

Control of Keratinocyte Proliferation and Differentiation by p63

The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKα and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.     

Keratinocyte Migration in the Developing Eyelid Requires LIMK2

In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.     

A Model Implicating the Langerhans Cell in Keratinocyte Proliferation Control

A tentative model is presented which is based on existing data and our own cell kinetic and morphological observations in mice. The model suggests that the epidermal Langerhans cell plays a role in proliferation control of keratinocytes and may also act as an epidermal stem cell.     

乳头瘤病毒11型完整基因组成功转染角质形成细胞并促其增殖

构建含人乳头瘤病毒11型(Human papillomavirus type 11, HPV11)完整开放读码框(Open reading frame, ORF)基因组的质粒并对其功能进行初步验证, 为构建HPV11转基因动物模型奠定基础。分别构建重组质粒pQE-Trisystem- EGFP/HPV11(pE/H)、pQE-Trisystem-EGFP/1.1copyHPV11(pE/1.1H), 将pE/1.1H、pE/H、闭环状HPV11基因组分别转染原代人角质形成细胞(Keratinocyte, KC)并进行检测。在质粒上成功构建了目的序列, 转染后在pE/1.1H组、pE/H组、HPV11组中检测到HPV11E6基因的表达; 在pE/1.1H组、pE/H组中检测到荧光; HPV11组、pE/H组、pE/1.1H组具备促细胞增殖功能, 实验组间比较pE/1.1H组稍弱(P<0.01), 但与对照组相比均差异极显著(P<0.01)。将具有完整ORF的HPV11基因组(1.1copyHPV11)成功构建于质粒上, 其具备野生型HPV11病理表型; 为研究低危型HPV致病机理提供了实验材料和方法学指导; 为进一步构建HPV11转基因小鼠奠定了基础。     

Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation.     

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.     

二咖啡酰奎宁酸对成纤维细胞增殖与功能的影响

探讨二咖啡酰奎宁酸（IBE5）对成纤维细胞活力,增殖及功能的影响。研究IBE5抗肝纤维化的机制,实验采用体外培养的NIH／3T3细胞作为成纤维细胞的替代模型,常规培养,质量深度为250,125,62.5,31.25,15.6,7.8mg/L的IBE5加入细胞中培养24h;[^3H]-TdR掺入法测定细胞增殖率;[^3H]-Pro掺入法测定细胞活力;[^3H]-Pro掺入,胶原酶消化法测定细胞内外胶原生成率,结果显示以上6个浓度的IBE5对细胞均无毒性,各组均可显著抑制细胞增殖,促进细胞活力,明显抑制胶原和透明质酸的合成,以250mg/L为最佳浓度,IBE5能显著抑制3T3细胞增殖,胶原和透明质酸合成并对细胞活力有促进作用,IBE5在 外具有显著的抗肝纤维化作用,对于防治肝纤维化可能有一定的临床意义和应用前景。     

Evaluation of Fibroblasts Adhesion and Proliferation on Alginate-Gelatin Crosslinked Hydrogel

Due to the relatively poor cell-material interaction of alginate hydrogel, alginate-gelatin crosslinked (ADA-GEL) hydrogel was synthesized through covalent crosslinking of alginate di-aldehyde (ADA) with gelatin that supported cell attachment, spreading and proliferation. This study highlights the evaluation of the physico-chemical properties of synthesized ADA-GEL hydrogels of different compositions compared to alginate in the form of films. Moreover, in vitro cell-material interaction on ADA-GEL hydrogels of different compositions compared to alginate was investigated by using normal human dermal fibroblasts. Viability, attachment, spreading and proliferation of fibroblasts were significantly increased on ADA-GEL hydrogels compared to alginate. Moreover, in vitro cytocompatibility of ADA-GEL hydrogels was found to be increased with increasing gelatin content. These findings indicate that ADA-GEL hydrogel is a promising material for the biomedical applications in tissue-engineering and regeneration.     

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis

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Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.     

脂肪干细胞与放射后成纤维细胞共培养模型的建立和lncRNA高通量测序

目的:探讨大鼠脂肪来源干细胞(adipose-derived stem cells,ADSCs)对放射后成纤维细胞(fibroblast,Fb)的影响。方法:体外原代培养SD大鼠ADSCs和真皮Fb,分别对两种细胞进行鉴定,Fb辐照后两种細胞建立共培养模型。将Fb分为3组,对其进行lncRNA和mRNA转录本高通量测序。结果:ADSCs高表达CD29和CD44,低表达CD31和CD45。大鼠Fb高表达波形蛋白。Fb单纯辐照和干細胞干预后转录水平有明显的差异。结论:ADSCs能使辐射后的Fb細胞lncRNA和mRNA转录水平明显的改变,提示多项生物学功能及信号通路在其中发挥作用。     

VILIP-1 Expression In Vivo Results in Decreased Mouse Skin Keratinocyte Proliferation and Tumor Development

VILIP-1, a member of the neuronal Ca²⁺ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.     

Cholesteatoma Fibroblasts Promote Epithelial Cell Proliferation through Overexpression of Epiregulin

To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial–mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.     

穿膜肽KGF-2的构建及其对HaCaT增殖作用的影响

目的:构建穿膜肽KGF-2(TAT-KGF-2),研究其对人角质形成细胞HaCaT的增殖作用。方法:采用普通PCR技术和特殊引物从人胚胎肺成纤维细胞cDNA中扩增出目的基因TAT-KGF-2,并将其插入pET-28a(+)载体中构建pET28a-TAT-KGF-2重组质粒;将该质粒转化大肠杆菌Rosetta(DE3),IPTG诱导表达重组蛋白TAT-KGF-2;通过Ni-NTA柱纯化获得目标重组蛋白并利用Western blot进行鉴定;MTT法研究TAT-KGF-2对HaCa的增殖作用。结果:成功构建pET28a-TAT-KGF-2重组质粒,经序列比对,与GenBank上公布的人KGF-2基因序列同源性达到100%;获得穿膜肽KGF-2蛋白,分子量约为19 kDa,纯度达到95%以上;TAT-KGF-2对HaCaT在12.5ng/ml时具有增殖作用,并呈浓度依赖性,而且与不具有穿膜功能的KGF-2相比有更强的增殖效果。结论:成功纯化出TAT-KGF-2蛋白,并验证其对HaCaT具有增殖作用,为进一步研究该蛋白的结构和功能奠定了基础。     

Polarity, Protrusion–Retraction Dynamics and Their Interplay during Keratinocyte Cell Migration

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 μg/cm² stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 μg/cm² fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 μg/cm² fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 μg/cm². The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 μg/cm² fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.