Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.     

TCDD exposure of human embryonic palatal shelves in organ culture alters the differentiation of medial epithelial cells

The highly toxic, polychlorinated aromatic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant throughout the environment. Epidemiology studies of populations accidentally exposed to TCDD have failed to identify TCDD as a human teratogen, but these studies are limited by the small numbers of exposed pregnancies and imprecise estimates of exposure. TCDD is highly teratogenic in mice, inducing cleft palate and hydronephrosis. TCDD exposure in vivo of embryonic mice alters the differentiation and expression of growth factors in the medial epithelial palatal cells. These alterations also occur in rat and mouse palates exposed to TCDD in organ culture. In the present study, human embryonic palatal shelves were cultured in the rodent organ culture system. In order to achieve in vitro the developmental stage at which fusion would normally occur, GD 52 shelves were cultured for 4 days, GD 53 shelves were cultured for 3 days, and GD 54 shelves were cultured for 3 days. Three of four palatal shelves exposed to 5 x 10(-11) M TCDD were identical to their homologous controls (right shelf cultured with control medium; left shelf cultured with TCDD-containing medium). TCDD at 1 x 10(-7) M produced cytotoxicity detected by transmission electron microscopy (TEM). Exposure to 1 x 10(-8) M TCDD resulted in continued incorporation of thymidine ([3H]-TdR detected autoradiographically) by palatal medial cells, failure of the medial peridermal cells to degenerate as observed by scanning electron microscopy (SEM), and differentiation into a stratified, squamous epithelium. These alterations are identical to those induced by TCDD in vitro in rat and mouse palatal cells. The main difference between these species is the level of TCDD required to elicit the responses. Cultured mouse palates respond to 5 x 10(-11) M TCDD with altered medial cell differentiation, and 1 x 10(-10) M TCDD is cytotoxic. The rat shelves respond with altered differentiation at 1 x 10(-8) M and cytotoxicity at 1 x 10(-7) M. All the human shelves respond at 1 x 10(-8) M TCDD with altered differentiation, 1 out of 4 responded at 5 x 10(-11) M, and cytotoxicity occurred at 1 x 10(-7) M. The present data suggest human embryonic palates are less sensitive than those of the C57BL/6N mouse, and that exposure to high levels of TCDD would be required to elicit altered differentiation in the palatal shelf.     

Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation

Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

TGF-alpha regulates TLR expression and function on epidermal keratinocytes

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-alpha, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-alpha regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human beta-defensins. These findings provide evidence that TGF-alpha up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.     

Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.     

Transforming growth factor-alpha

In summary, although TGF-alpha was initially found in tumors, a number of later studies, some of them from the author's laboratory, have shown that TGF-alpha should no longer be considered a tumor associated growth factor. Rather, TGF-alpha is a normal physiological ligand for the EGF receptor. Table 2 lists some of the normal cellular sources of TGF-alpha. Our list is incomplete, but we know that TGF-alpha is made in keratinocytes and a number of epithelial cells, including gut and breast epithelial cells. It seems very likely that TGF-alpha is a major growth factor secreted by cells of epithelial origin. Zena Werb's and Russell Ross's groups have shown that activated macrophages make TGF-alpha. We have shown that brain makes TGF-alpha and Jeff Kudlow has found TGF-alpha made in the pituitary. Data from several sources, including David Lee, the author's laboratory, and Zena Werb's laboratory has shown that TGF-alpha is made during embryonic development. Therefore, it is now important to look at TGF-alpha in its normal physiological context. Finally, it should be stressed that, as was mentioned above, TGF-alpha is not necessarily a secreted growth factor 50 amino acids long. There is quite a bit of processing of the larger precursor that may or may not take place. This processing, which determines the ultimate size and location of the molecule, is also likely to influence its physiological action.     

TGF-alpha increases human mesenchymal stem cell-secreted VEGF by MEK- and PI3-K- but not JNK- or ERK-dependent mechanisms

Transforming growth factor-alpha (TGF-alpha) may be an important mediator of wound healing and the injury response. Human bone marrow mesenchymal stem cells (MSCs) release VEGF as a potentially beneficial paracrine response; however, it remains unknown whether TGF-alpha stimulates the production of VEGF from MSCs and, if so, by which mechanisms. We hypothesized that TGF-alpha would increase human MSC VEGF production by MAP kinase kinase (MAPKK/MEK), phosphatidylinositol 3-kinase (PI3-K)-, ERK, and JNK-dependent mechanisms. To study this, MSCs were cultured and divided into the following groups: 1) with vehicle; 2) with various stimulants alone: TGF-alpha, TNF-alpha, or TGF-alpha+TNF-alpha; 3) with individual kinase inhibitors alone (two different inhibitors for each of the following kinases: MEK, PI3-K, ERK, or JNK); and 4) with the above stimulants and each of the eight inhibitors. After 24-h incubation, a TGF-alpha dose-response curve demonstrated that low-dose TGF-alpha (500 pg/ml) suppressed MSC production of VEGF compared with vehicle (502 +/- 16 pg/10(5) cells/ml to 332 +/- 9 pg/10(5) cells/ml), while high-dose TGF-alpha (250 ng/ml) significantly increased MSC VEGF production (603 +/- 24 pg/10(5) cells/ml). High-dose TGF-alpha also increased TNF-alpha-stimulated release of VEGF from MSCs. MSCs exposed to TGF-alpha and/or TNF-alpha also demonstrated increased activation of PI3-K, JNK, and ERK. The TGF-alpha-stimulated production of VEGF by MSCs and the additive effect of TNF-alpha and TGF-alpha on VEGF production were abolished by MEK and PI3-K inhibition, but not ERK or JNK inhibition. Our data suggest that TGF-alpha increases VEGF production in MSCs via MEK- and PI3-K- but not ERK- or JNK-dependent mechanisms.     

Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal keratinocytes

In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-alpha (TGF-alpha); transforming growth factor-beta (TGF-beta) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-alpha which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-beta, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells.     

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.     

Induction of transforming growth factor-alpha in activated human alveolar macrophages

The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-alpha (TGF-alpha) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-alpha. Two different molecular species of TGF-alpha activity (approximately 8,500-12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-alpha. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.     

EGF and TGF-alpha in wound healing and repair

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.     

Generation of transforming growth factor-alpha from the cell surface by an O-glycosylation-independent multistep process

The precursor for transforming growth factor-alpha (TGF-alpha) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-alpha receptors on adjacent cells or can be cleaved to release TGF-alpha that diffuses into the medium. Cleavage of pro-TGF-alpha occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-alpha sequence. To characterize the cleavage process of pro-TGF-alpha and the role of glycosylation in this process, we have introduced a pro-TGF-alpha expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-alpha sequence and the cytoplasmic pro-TGF-alpha C-terminal domain shows that cleavage of pro-TGF-alpha in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-alpha reaches the cell surface. In the first step, pro-TGF-alpha rapidly (t1/2 = 30 min) loses the amino-terminal segment that precedes the TGF-alpha sequence. In the second step, pro-TGF-alpha is cleaved at the carboxyl terminus of the TGF-alpha sequence releasing this factor into the medium. This second step is slow (t1/2 = 2 h). The action of pancreatic elastase added to CHO-TGF-alpha cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-alpha in ldlD-TGF-alpha cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-alpha may be processed with different kinetics which can lead to retention of pro-TGF-alpha on the cell surface.