Epidermal morphogenesis in an <Emphasis Type="BoldItalic">in-vitro</Emphasis> model using a fibroblasts-embedded collagen scaffold

Summary A novel culture system included a self-designed bi-layer 3-D collagen scaffold with different pore size on both sides and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-β1), in the cell–cell and cell–matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air–liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10 ng per ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10 ng per ml of EGF addition in the air-lifted stage resulted in uneven cell–matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-β1).     

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.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Effects of platelet-contained growth factors (PDGF, EGF, IGF-I, and TGF-β) on DNA synthesis in porcine aortic smooth muscle cells in culture

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-β (TGF-β) are potent mitogens present in human platelets. Since they are likely to be released simultaneously at the site of vessel injury, their combined effects on vascular smooth muscle cells are more relevant physiologically than their individual actions. Therefore, we added various concentrations of growth factors to quiescent porcine aortic smooth muscle cells cultured in lowserum (0.5%) medium and measured the amount of [³H]thymidine incorporated into DNA. Effect of TGF-β alone was concentration-dependent: stimulatory (1.5-fold increase over the basal) at 0.025 ng/ml and inhibitory at 0.1 ng/ml. Effects of the other three growth factors on DNA synthesis were only stimulatory; their maximally effective concentrations were 20 ng/ml for PDGF (eightfold over the basal), 40 ng/ml for EGF (sixfold increase), and 20 ng/ml for IGF-I (fourfold increase). When PDGF, EGF, and IGF-I were added at submaximally effective concentrations, their effects were additive. TGF-β at 1 ng/ml inhibited at least 50% of the effects of 20 ng/ml EGF and of 10 ng/ml IGF-I, whereas inhibition of the effect of 10 ng/ml PDGF required 10 ng/ml of TGF-β. The concentration of TGF-β needed to inhibit 50% of the combined effect of EGF, IGF-1, and PDGF was 5 ng/ml. These results show complex interrelationships between the growth factors contained in the α-granules of human platelets in their effects on porcine aortic smooth muscle cells.     

Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium

A serum-free medium (LEP-1) has been developed for mouse epidermal keratinocytes. LEP-1 consists of "Ca2+-free" Eagle's MEM with non-essential amino acids and seven added supplements (transferrin, 5 micrograms/ml; epidermal growth factor (EGF), 5 ng/ml; hydrocortisone, 0.5 microM; insulin, 5 micrograms/ml; phosphoethanolamine and ethanolamine, each 50 microM; bovine pituitary extract, 180 micrograms of protein/ml). Although serum-free the culture system was dependent for growth on bovine pituitary extract as the only still undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was developed to investigate the action of growth factors, hormones and other supplements on keratinocytes. Cells grown in LEP-1 (calcium concentration was 0.03 mM) maintained a high proliferative rate and presented the typical morphology of basal epidermal cells. When the calcium concentration of the medium was raised to 1.0 mM, the cells were triggered to differentiate terminally. The epithelial nature of the cells was demonstrated both by electron microscopy and by immunostaining with anti-keratin antibody. The maturation stage of the keratinocytes was defined by several morphological features during the proliferative phase and in terminally differentiating cultures. This serum-free system supported a useful number of cell divisions while keratinocytes retained the capacity to undergo terminal differentiation when given the appropriate stimulus. It provides, therefore, provides a useful model for investigations on growth, differentiation and malignant transformation of epidermal cells in culture.     

Human keratinocytes maintain reversible anti-apoptotic defenses in vivo and in vitro

Human keratinocytes proliferate and differentiate in an epidermal environment where induction of apoptosis can be triggered by ultraviolet radiation (UVR), activated lymphocytes and cytokines. The purpose of this study was to determine whether keratinocytes were susceptible to apoptosis induced by ionophore, ultra-violet radiation, cytokines or crosslinking of CD95 (Fas/APO-1). In normal human skin exposed to two minimal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apoptotic cells were identified throughout the mid to upper epidermis. However, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epidermis. Human keratinocytes and keratinocyte cell lines cultured in vitro developed maximal apoptosis 48 h after radiation. Human keratinocytes cultured in full growth factor supplements were resistant to UVR-induced apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were completely resistant to apoptosis induced by interferon-, interferon-, IL-2, IL-6, TNF-, IL-1Ra, and GM-CSF. A subset of the cells in cultures of keratinocytes and transformed keratinocyte cell lines died by apoptosis in response to anti-Fas, IL-1 and TNF- plus IFN- and ionophore. Second passage freshly isolated human keratinocytes were much more resistant to apoptosis induced by ionophore, anti-Fas and cytokines than were transformed keratinocyte cell lines. Calcium shift to induce differentiation in second-passage keratinocyte cultures made keratino-cytes even more resistant to UVR-induced apoptosis. This parallels the lack of UVR-induced apoptosis observed in the most differentiated keratinocytes in irradiated human skin. Both keratinocytes and kerati-nocyte cell lines express rather low levels of the anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis-resistant cell types. The differences between keratinocytes and keratinocyte cell lines in suscepti-bility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of growth factors from keratinocytes decreased cell survival following UVR and increased the induction of apoptosis. Inhibition of protein synthesis with cyclo-heximide also made keratinocytes more susceptible to UVR-induced apoptosis, indicating that anti-apop-totic defences in cultured keratinocytes are dependent on active protein synthesis. These experiments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiation and growth factor withdrawal.     

BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.     

Transforming growth factor type β is a specific inhibitor of 3T3 T mesenchymal stem cell differentiation

In the current studies we examined the effects of transforming growth factor type β (TGF-β) on the control of differentiation of BALB/c 3T3 T stem cells. We report that TGF-β is a potent, reversible inhibitor of adipocyte differentiation (50% inhibition at ˜0.06–0.08 ng/ml), while other biologically active polypeptides, such as epidermal growth factor (EGF), human growth hormone (hGH), and somatomedin C, have no specific effect on differentiation at even higher concentrations (200 ng/ml). We also report that TGF-β inhibits differentiation in a cell cycle-dependent manner by its effect on a specific phase in the differentiation process. We therefore suggest that if TGF-β is an important regulatory factor, one of its critical mechanisms of action may be its ability to inhibit the process of cell differentiation.     

Cell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation

Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G₀) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca²⁺ Dulbecco's modified Eagle's medium/F12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14 h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse-labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16 h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.     

Modulation of TGF-β-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

Keratinocytes migrating from a wound edge or initiating malignant invasion greatly increase their expression of the basement membrane protein Laminin-322 (Lam332). In culture, keratinocytes initiate sustained directional hypermotility when plated onto an incompletely processed form of Lam332 (Lam332′) or when treated with transforming growth factor beta (TGF-β), an inducer of Lam332 expression. The development and tissue architecture of stratified squamous and prostate epithelia are very different, yet the basal cells of both express p63, α6β4 integrin, and Lam332. Keratinocytes and prostate epithelial cells grow well in nutritionally optimized culture media with pituitary extract and certain mitogens. We report that prostate epithelial cells display hypermotility responses indistinguishable from those of keratinocytes. Several culture medium variables attenuated TGF-β-induced hypermotility, including Ca⁺⁺, serum, and some pituitary extract preparations, without impairing growth, TGF-β growth inhibition, or hypermotility on Lam322′. Distinct from its role as a mitogen, EGF proved to be a required cofactor for TGF-β-induced hypermotility and could not be replaced by HGF or KGF. Prostate epithelial cells have a short replicative lifespan, restricted both by p16^INK4A and telomere-related mechanisms. We immortalized the normal prostate epithelial cell line HPrE-1 by transduction to express bmi1 and TERT. Prostate epithelial cells lose expression of p63, β4 integrin, and Lam332 when they transform to invasive carcinoma. In contrast, HPrE-1/bmi1/TERT cells retained expression of these proteins and normal TGF-β signaling and hypermotility for >100 doublings. Thus, keratinocytes and prostate epithelial cells possess common hypermotility and senescence mechanisms and immortalized prostate cell lines can be engineered using defined methods to yield cells retaining normal properties.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

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.     

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. I. Effect of liposome B components on colony growth

Summary An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64–24 with far less protein supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 μg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 μg/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 μg/ml liposome B (a mixture of soy lecithin, sphingomyelin, cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine or sphingomyelin. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by B.A.VdH. in partial fulfillment of the requirements for the Ph.D. degree. This research was supported by grant CA-15305 to R.G.H. and grant CA-30545 to T.K.-S., both from the National Institutes of Health, Bethesda, MD.     

EGF receptor crosstalks with cytokine receptors leading to the activation of c-Jun kinase in response to UV irradiation in human keratinocytes

Ultraviolet (UV) irradiation causes photoageing through induction of matrix-degrading metalloproteinases (MMP), which are upregulated by activator protein-1 (AP-1) (Jun/Fos). The c-Jun kinase activity proves to be critically important in the regulation of AP-1 activity. Our previous studies showed that UV irradiation activates epidermal growth factor receptor (EGFR) and cytokine receptors leading to the activation of c-Jun kinase in cultured human skin keratinocytes in vitro and in human skin in vivo. However, the mechanism of UV-induced cell surface receptor activation and the crosstalk among growth factor receptor and cytokine receptors were not fully investigated. This study showed that UV (30 mJ/cm(2))-induced EGFR tyrosine phosphorylation in a manner similar to EGF (100 ng/ml), or IL-1beta (10 ng/ml) in cultured human keratinocytes. In all cases, EGFR tyrosine phosphorylation was completely inhibited by pretreatment of PD153035 (100 nM, 1 h). Also observed was that UV induced autophosphorylation of interleukin 1 receptor associated kinase (IRAK) in a manner analogous to IL-1beta or EGF. In both UV and EGF cases, the phosphorylation of IRAK was inhibited by pretreatment of PD153035. However, IL-1beta-induced IRAK activation was not affected by PD153035. In vitro kinase assay using GST-c-Jun as a substrate revealed that pretreatment of PD153035 completely inhibited UV- and IL-1-induced c-Jun kinase activity in cultured keratinocytes. Taken together, the above data suggest that EGFR plays dominant role in the crosstalk among growth factor receptor and cytokine receptors leading to the activation of c-Jun kinase upon UV irradiation, and that EGFR could be one of the targets for clinical and cosmetical prevention of UV-induced skin aging.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Expression of intermediate filaments,EGF and TGF-α in early human kidney development

The spatial and temporal expression patterns of cytokeratins, vimentin, epithelial growth factor (EGF) and transforming growth factor alpha (TGF-α), were investigated in the 5–9-week old human mesonephros and metanephros. Vimentin was found in all mesonephric structures, while cytokeratins were seen only in the mesonephric tubules. EGF and TGF-α were detected early in all mesonephric structures, and immunoreactivity to both factors decreased in later stages. In the 5–6-week metanephros, vimentin immunoreactivity was found in all structures and later increased in the collecting system and interstitium. In the 5th week, cytokeratins 8 and 19 appeared in the ureteric bud and ampullae, and later showed increasing immunoreactivity in the collecting system and nephrons. The coexpression of intermediate filament proteins in metanephric development is a temporary feature and might be associated with mesenchymal to epithelial transformation of developing nephrons. In adult kidneys, such coexpression is associated with fibrosis or carcinomatous changes. At early stages, immunoreactivity to EGF and TGF-α was detected in all metanephric structures and from the 7th week onward, it decreased in differentiating nephrons. EGF and TGF-α patterns of appearance indicate their role in induction, proliferation and growth of metanephric structures. Disturbances in that pattern might cause reduction in kidney growth.     

Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins

Summary Rat liver epithelial cells are induced to migrate by epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) in serum-free medium supplemented with insulin. Immunohistological staining of the migration tracks containing laminin and fibronectin has allowed a quantitative analysis of the process. The growth factor-induced migration is relatively slow, but very efficient. Between 24 and 48 h after exposure to EGF (or TGF-α), 50 to 70% of the cells have migrated away from their site of initial attachment and spreading. This delayed effect of the interaction of the receptor with its ligands is associated with changes in gene expression, but is not associated with a stimulation of cell proliferation. In serum-free medium supplemented with insulin, the cells secrete six major proteins, as revealed by SDS-polyacrylamide gel electrophoresis. The media of cultures supplemented with insulin plus EGF (or TGF-α) contain in addition two new proteins and an increased amount of fibronectin. One secreted protein is synthesized in significantly reduced amounts. The most conspicuously EGF-induced protein (EIP-1, Mr 47 000) is detected within 2 h, depends on the continued presence of the growth factor, and has not been detected as bound to the substratum. The stringent regulation of EIP-1 suggests that this gene product might participate in the modulation of the changes induced by the growth factor. The system is being used for the further analysis of the regulation of gene expression by EGF and of the migration of normal and neoplastically transformed epithelial cells. This paper is dedicated to the memory of Dr. Luis F. Leloir. A preliminary communication has been presented at the Cold Spring Harbor Meeting on Liver Gene Expression, May 1987. Editor's Statement Mitogen-stimulated gnees are an active area of study with fibroblastic systems. In this paper the approach is extended to epithelial cells and functional correlations are also made.