角质细胞生长因子研究进展

角质细胞生长因子（KGF）属于成纤维细胞生长因子家族,它通过与受体（KGFR）的结合,特异性地刺激上皮细胞的增殖.KGF基因表达受到正负调控作用,正负调控作用的平衡对于KGF正常发挥功能具有重要意义.KGF具有多种生物学功能：参与组织、器官的发育,具有损伤防治功能,与癌症的发生有着密切的联系.     

Lens-Specific Expression of PDGF-A Alters Lens Growth and Development

The vertebrate lens provides anin vivomodel to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development byin situhybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-α receptor (PDGF-αR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-αR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens developmentin vivo,we generated transgenic mice that express human PDGF-A in the lens under the control of the αA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific β-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cellsin vivo.Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Elevated TGFbeta signaling inhibits ocular vascular development

Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.     

Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. Synthesized by cells of the dermal component of skin, KGF's potent mitogenic activity is on the epidermal component, which harbors the receptors for this factor. To explore the possible role of KGF in mesenchymal-epithelial interactions in skin, we used a human keratin 14 promoter to target expression of human KGF cDNA to the stratified squamous epithelia of transgenic mice. Mice expressing KGF in their epidermis typically appeared frail and weak, and often had grossly wrinkled skin. These mice exhibited a gross increase in epidermal thickness accompanied by alterations in epidermal growth and differentiation. Most remarkably, animals displayed several striking and unexpected changes, including a marked suppression of hair follicle morphogenesis and suppression of adipogenesis. With age, some animals developed gross transformations in the tongue epithelium and in epidermis. In addition, they exhibited elevated salivation and their salivary glands showed signs of altered differentiation. Collectively, our findings provide new and important insights into the roles of KGF, implicating this potent growth factor in eliciting global effects not only on growth, but also on development and differentiation, of skin and other tissues. In particular, KGF seems to interfere with signalling of some mesenchymal-epithelial interactions.     

Cholesteatoma-associated fibroblasts modulate epithelial growth and differentiation through KGF/FGF7 secretion

The keratinocyte growth factor (KGF/FGF7), produced by stromal cells, is a key paracrine mediator of epithelial proliferation, differentiation and migration. Expression of the growth factor is increased in wound healing and in hyperproliferative epithelial diseases, as a consequence of the activation of dermal fibroblasts by the inflammatory microenvironment. The middle ear cholesteatoma, an aural epidermal pathology characterized by keratinocyte hyperproliferation and chronic inflammation, may represent a model condition to study the epithelial-mesenchymal interactions. To develop an in vitro model for this disease, we isolated and characterized human primary cultures of fibroblasts associated with the cholesteatoma lesion, analyzing their secretory behaviour and degree of differentiation or activation. Compared to the perilesional or control normal fibroblasts, all cultures derived from cholesteatoma tissues were less proliferating and more differentiated and their highly variable activated phenotype correlated with the secretion of KGF as well as of metalloproteases 2 and 9. Culture supernatants collected from the cholesteatoma-associated fibroblasts were able to increase the proliferation and differentiation of human keratinocytes assessed by the expression of Ki67 and keratin-1 markers. The single crucial contribution of the KGF released by fibroblasts on the keratinocyte biological response was shown by the specific, although partial, block induced by inhibiting the KGF receptor or by immunoneutralizing the growth factor. Altogether, these results suggest that the activation of the stromal fibroblasts present in the pathological tissue, and the consequent increased secretion of KGF, play a crucial role in the deregulation of the epidermal proliferation and differentiation.     

Heparin-binding keratinocyte growth factor is a candidate stromal-to-epithelial-cell andromedin.

The growth of isolated epithelial and stromal cells from both androgen-dependent normal rat prostate and an androgen-responsive model rat prostate tumor is androgen-independent. When added to co-cultures of epithelial and stromal cells separated by a semipermeable membrane, androgen stimulated epithelial cell growth without an effect on stromal cell growth. Northern blot and nuclease protection analysis of mRNA revealed that stromal cells specifically expressed an androgen-sensitive secreted member of the heparin-binding fibroblast growth factor family [keratinocyte growth factor (KGF)/fibroblast growth factor-7]. KGF was mitogenic for epithelial cells, but not for stromal cells. Epithelial cells expressed specifically a splice variant of the bek receptor gene that specifically binds KGF. Expression of the bek receptor gene in stromal cells was undetectable by Northern blot and nuclease protection analyses. The results suggest that stromal cell-derived KGF has the properties of an andromedin, which mediates the indirect control of epithelial cell proliferation by androgen through a directional stromal-to-epithelial cell paracrine mechanism.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.     

Attenuation and loss of hormonal modulation of KGF (FGF-7)/KGF receptor expression and mitogenesis during mammary tumor progression

Keratinocyte growth factor (KGF), alone and in synergism with progesterone (P) and prolactin (PRL), is mitogenic for normal mammary epithelium (ME) in vitro. In addition, P can upregulate ME sensitivity to KGF by slowing KGF receptor (KGFR) mRNA turnover in vitro. These hormonal interactions with KGF in vitro raise the possibility that alterations in these interactions can play a role in hormone-dependent mammary tumor growth and progression. The effect of hormones on KGF mitogenesis and the regulation of KGFR expression was examined in pregnancy-dependent (PDT) and ovarian-independent (OIT) mouse mammary tumors. In serum-free, collagen gel cell culture, dose/response (2-20 ng/ml) and time course studies showed that KGF stimulated the proliferation of PDT (not OIT) cells but synergism with P or PRL was not observed. The level of KGFR mRNA in PDT cells was not significantly different from normal ME but in OIT it was reduced more than 90%. P did not affect KGFR mRNA turnover in cultured PDT cells. However, KGFR mRNA was more stable in PDT cells compared to normal ME; after 6 days culture in basal medium, KGFR mRNA levels declined 40% vs. 85% previously shown for normal ME. Determination of KGF mRNA levels in tissues showed that it was lower in PDT compared to normal mammary gland and not detectable in OIT. These data show that in PDT both KGF-stimulated mitogenesis and the regulation of KGFR expression are independent of hormones. OIT has progressed to independence from any KGF influence. Thus, a subset of hormonally regulated pathways related to epithelial/stromal cell interactions can be lost in hormone-dependent mammary tumors during tumor progression.     

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

Hepatocyte growth factor and keratinocyte growth factor enhance IL-1-induced IL-8 secretion through different mechanisms in Caco-2 epithelial cells

A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.     

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.