共查询到20条相似文献,搜索用时 15 毫秒
1.
Hannen RF Michael AE Jaulim A Bhogal R Burrin JM Philpott MP 《Biochemical and biophysical research communications》2011,(1):62-67
Mechanical stretch has been shown to increase vascular endothelial growth factor (VEGF) expression in cultured myocytes. Sympathetic neurons (SN) also possess the ability to express and secrete VEGF, which is mediated by the NGF/TrkA signaling pathway. Recently, we demonstrated that SN respond to stretch with an upregulation of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF). Whether stretch increases neuronal VEGF expression still remains to be clarified. Therefore, SN from the superior cervical ganglia of neonatal Sprangue Dawley rats were exposed to a gradual increase of stretch from 3% up to 13% within 3 days (3%, 7% and 13%). Under these conditions, the expression and secretion of VEGF was analyzed. Mechanical stretch significantly increased VEGF mRNA and protein expression (mRNA: control = 1 vs. stretch = 3.1; n = 3/protein: control = 1 vs. stretch = 2.7; n = 3). ELISA experiments to asses VEGF content in the cell culture supernatant showed a time and dose dependency in VEGF increment due to stretch. NGF and CNTF neutralization decreased stretch-induced VEGF augmentation in a significant manner. This response was mediated in part by TrkA receptor activation. The stretch-induced VEGF upregulation was accompanied by an increase in HIF-1α expression. KDR levels remained unchanged under conditions of stretch, but showed a significant increase due to NGF neutralization. In summary, SN respond to stretch with an upregulation of VEGF, which is mediated by the NGF/CNTF and TrkA signaling pathway paralleled by HIF-1α expression. NGF signaling seems to play an important role in regulating neuronal KDR expression. 相似文献
2.
CRH cutaneous expression is significantly enhanced after exposure to various stimuli (Physiol Rev 2000, 80;979-1020). We evaluated the effect of CRH on cytokine production in HaCaT keratinocytes, a cell line shown to express CRH receptors coupled to cAMP activation and calcium-dependent transmission pathways. It is demonstrated for the first time that exogenously added CRH stimulates production of IL-6 and IL-11. It also inhibits production of IL-1beta and does not affect TNF-alpha production. Our results indicate that CRH function(s) during cutaneous stress may be mediated by differential effects on cytokine production. 相似文献
3.
Alt A Gartsbein M Ohba M Kuroki T Tennenbaum T 《Biochemical and biophysical research communications》2004,314(1):17-23
In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation. 相似文献
4.
Zhu Z Hotchkiss SA Boobis AR Edwards RJ 《Biochemical and biophysical research communications》2002,297(1):65-70
The complement and level of expression of P450 enzymes in male Fischer F344 rat whole skin and cultured keratinocytes were investigated using a panel of mono-specific antibodies. In whole skin microsomal fraction, immunoreactive bands corresponding to CYP2B12, CYP2C13, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP3A2 were detected whereas CYP1A1, CYP1A2, and CYP2C12 were absent. Skin levels were all between 0.1% and 4.7% of those found in liver, except for CYP2D4, which was not detected in liver. Keratinocytes were isolated from rat skin, cultured for up to 42 days, and changes in the levels of CYP3A1, CYP3A2, and CYP2E1 determined. Levels were low in isolated keratinocytes, but they increased markedly in culture, reaching a maximum at 10-14 days, where they were similar to those found in fresh skin. This suggests that primary keratinocytes grown in culture for 10-14 days may provide a useful experimental model to study P450-catalysed metabolism of xenobiotics in skin. 相似文献
5.
Hussein MR 《Cell biology international》2007,31(7):748-751
KIT proto-oncogene is a receptor tyrosine kinase critical for signal transduction in several cell types. An understanding of KIT distribution in normal skin may reveal new insights into its implication in cutaneous biology and its associated disease states. This study examines expression patterns of KIT protein in normal human skin. KIT protein expression was examined in 50 specimens of normal human skin using specific antibodies (CD117) and immunoperoxidase staining methods. KIT protein expression was seen in keratinocytes (stratum basale), melanocytes, mast cells, and in both sebaceous and sweat glands. In contrast, KIT protein expression was absent in stratum spinosum, stratum granulosum, stratum corneum, blood vessels and arrector pili muscle. Thus, the expression of KIT protein in normal human skin suggests its possible role in regulation of cutaneous development and function. 相似文献
6.
Zeitvogel J Dalpke A Eiz-Vesper B Kracht M Dittrich-Breiholz O Werfel T Wittmann M 《The Journal of biological chemistry》2012,287(13):9923-9930
Suppressor of cytokine signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a deregulated immune response in chronic inflammatory skin diseases. Thus, it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. Because we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes compared with autologous blood-derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3-activating cytokines investigated, including IL-6, IL-22, and oncostatin M, also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell type-specific impaired capacity to up-regulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases. 相似文献
7.
Mª Fátima Guarizo Klingbeil Marisa Roma Herson Elier Broche Cristo Décio dos Santos Pinto Jr Daniele Yoshito Monica Beatriz Mathor 《Cell and tissue banking》2009,10(3):197-204
The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical,
resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of
the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of
the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used.
This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The
results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar
restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining
time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia. 相似文献
8.
9.
Gagnoux-Palacios L Hervouet C Spirito F Roques S Mezzina M Danos O Meneguzzi G 《The journal of gene medicine》2005,7(9):1178-1186
BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes. 相似文献
10.
Kakizaki I Itano N Kimata K Hanada K Kon A Yamaguchi M Takahashi T Takagaki K 《Archives of biochemistry and biophysics》2008,471(1):85-93
Hyaluronan controls keratinocyte proliferation and regeneration. We examined effect of UV on the expression of hyaluronan synthases (HASs) and hyaluronidases in cultured normal human newborn foreskin epidermal keratinocytes, NHEK(F). HAS3 mRNA was expressed predominantly and HAS2 mRNA expressed in lesser amounts and both were up-regulated after a single irradiation with moderate UVB but hyaluronidases was unchanged. Increased accumulation of hyaluronan in the culture medium mirrored the UVB-induced increase in the mRNA levels of HAS3 and HAS2. Unexpectedly, hyaluronan derived from UVB-irradiated and non-irradiated cells had identical size distribution. Increased expression of KGF and IL-1β was detected just prior to the increase of HAS3 and HAS2 mRNAs after UVB irradiation. Antibody-neutralization study revealed that KGF and/or IL-1β were at least involved in the up-regulation of HAS3 and HAS2 expressions. UVB-irradiated cells may enhance hyaluronan production to maintain homeostasis through up-regulation of HAS3 and HAS2 genes via cytokine response mechanism. 相似文献
11.
The silk protein sericin has been identified as a potent antioxidant and photoprotective agent against ultraviolet B (UVB)
irradiation in mouse skin model. In this study, we have investigated the anti-apoptotic effect of sericin in UVB (30 mJ/cm2)-irradiated human keratinocytes. Flow cytometry analysis has shown that pre-treatment with sericin inhibits UVB-induced apoptosis.
The pre-treatment with sericin suppresses bax expression, up-regulates the expression of bcl-2, prevents both the activation
of caspase-3 and cleavage of Poly (ADP-ribose) polymerase. Generation of intracellular hydrogen peroxide in UVB-treated keratinocytes
is inhibited through pre-treatment with sericin suggesting that sericin probably prevents mitochondrial damage.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes 总被引:1,自引:0,他引:1
MiRan Seo 《Biochemical and biophysical research communications》2010,393(2):190-2230
Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the α subunit of Gq protein (Gαq) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active Gαq (GαqQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of Gαq with shRNA in HaCaT human keratinocytes. Gαq was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase Cδ (PKCδ), and matrix metaloprotease-2 (MMP-2). Moreover, GαqQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that Gαq mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKCδ and MMP-2 in HaCaT cells. 相似文献
13.
Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can
be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium.
Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of
viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation
(basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although
they do not fully substitute for BPE. 相似文献
14.
Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes 总被引:10,自引:0,他引:10
Büth H Luigi Buttigieg P Ostafe R Rehders M Dannenmann SR Schaschke N Stark HJ Boukamp P Brix K 《European journal of cell biology》2007,86(11-12):747-761
Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration. 相似文献
15.
Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support. 相似文献
16.
Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7 总被引:3,自引:0,他引:3
Smith L Dahler AL Cavanagh LL Popa C Barnes LM Serewko-Auret MM Fai Wong C Saunders NA 《Experimental cell research》2004,294(2):356-365
We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation. 相似文献
17.
A mathematical model of mitotic activity of epidermis in normal skin and skin afflicted with psoriasis is presented as a system of two autonomous nonlinear differential equations. Its qualitative analysis was carried out and numerical solutions were obtained at the parameter values corresponding to these states. It was shown that in the norm, a single stable equilibrium of a "focus" type exists in the system; whereas in psoriasis, owing to an increase in the growing fraction, hyperproliferation, and enhanced migration of interacting keratinocytes, a stable limit cycle arises from the state of unstable focus. In this paper we also report on the results of computer modeling of synchronization of self-excited oscillations of keratinocyte population density in psoriatic lesions by an external periodic force. This synchronization is viewed as a possible mechanism of the clinically observed dependence of psoriasis course on some natural factors of cyclic nature. 相似文献
18.
Cdc42 is a small GTPase, which acts as a molecular switch to regulate a wide variety of cellular functions, such as actin cytoskeleton organization, cell proliferation, apoptosis, cell migration and in particular, cell polarity. Formation and maintenance of the basement membrane is a polarized process, which requires directed secretion, deposition and organization of basement membrane components at the basal side of epithelial cells. In the current study, we analyzed the maintenance of skin basement membrane in mice with a keratinocyte-restricted deletion of the Cdc42 gene. In the absence of Cdc42, basement membrane components became aberrantly deposited and the processing of laminin 5 was impaired in parts of the dermal-epidermal junction. These impairments became more severe with age and corresponded to local defects of the basement membrane in 4.5-month-old mutant mice. However, both, structure and number of hemidesomosomes were not significantly changed in the Cdc42 mutant skin compared with the control mice and no blister formation was observed in mutant skin. These data indicate that Cdc42 in keratinocytes is important for maintenance of the basement membrane of skin. 相似文献
19.
Schebb NH Buchholz BA Hammock BD Rice RH 《Journal of biochemical and molecular toxicology》2012,26(6):230-234
Previous studies of triclocarban suggest that its biotransformation could yield reactive metabolites that form protein adducts. Since the skin is the major route of triclocarban exposure, present work examined this possibility in cultured human keratinocytes. The results provide evidence for considerable biotransformation and protein adduct formation when cytochrome P450 activity is induced in the cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a model Ah receptor ligand. Since detecting low adduct levels in cells and tissues is difficult, we utilized the novel approach of accelerator mass spectrometry for this purpose. Exploiting the sensitivity of the method, we demonstrated that a substantial portion of triclocarban forms adducts with keratinocyte protein under the P450 inducing conditions employed. 相似文献
20.
We show that a single low-dose exposure of human epidermal keratinocytes (NHEK) to an FS20 light source in vitro can induce the formation of mitochondrial DNA deletions in a PCR detection assay. We used primer sets specifically designed to exclude amplification of segments containing the common deletion, but which could detect possibly lower abundance deletions generated within the same region of the mitochondrial genome. We characterized eight novel deletions of which six were generated from cut sites within, or adjacent to, short direct repeats. Two deletions involved cut sites in inverted tetrameric repeats; one of these also involved an insertion. 相似文献