Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Role of phospholipase Cgamma-induced activation of protein kinase Cepsilon (PKCepsilon) and PKCbetaI in epidermal growth factor-mediated protection of tight junctions from acetaldehyde in Caco-2 cell monolayers

Epidermal growth factor (EGF) protects the intestinal epithelial tight junctions from acetaldehyde-induced insult. The role of phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) isoforms in the mechanism of EGF-mediated protection of tight junction from acetaldehyde was evaluated in Caco-2 cell monolayers. EGF-mediated prevention of acetaldehyde-induced decrease in transepithelial electrical resistance and an increase in inulin permeability, and subcellular redistribution of occludin and ZO-1 was attenuated by reduced expression of PLCgamma1 by short hairpin RNA. EGF induced a rapid activation of PLCgamma1 and PLC-dependent membrane translocation of PKCepsilon and PKCbetaI. Inhibition of PKC activity or selective interference of membrane translocation of PKCepsilon and PKCbetaI by RACK interference peptides attenuated EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. BAPTA-AM and thapsigargin blocked EGF-induced membrane translocation of PKCbetaI and attenuated EGF-mediated prevention of acetaldehyde-induced disruption of tight junctions. EGF-induced translocation of PKCepsilon and PKCbetaI was associated with organization of F-actin near the perijunctional region. This study shows that PLCgamma-mediated activation of PKCepsilon and PKCbetaI and intracellular calcium is involved in EGF-mediated protection of tight junctions from acetaldehyde-induced insult.     

Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.     

Protein kinase C regulates the phosphorylation and cellular localization of occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.     

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.     

Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.     

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ‐mediated phosphorylation of occludin

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)‐1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP‐activated protein kinase (AMPK) and PKCζ in metformin‐induced effects. Pretreatment with metformin prevented the S. aureus‐induced changes in ZO‐1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl‐coA‐carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK‐PKCζ pathway.     

ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.     

Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity

At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.     

Dynamic changes in protein components of the tight junction during liver regeneration

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.     

Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near the zonula occludens complex. Antibodies against α-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, Gα₁₂ and PKC ζ, were the only isoforms of these proteins present at the cell borders. In subconfluent monolayers, Gα₁₂ and PKC ζ were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that Gα₁₂ and PKC ζ may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions. Received: 29 July 1995/Revised: 13 October 1995     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

TPA-induced signal transduction: a link between PKC and EGFR signaling modulates the assembly of intercellular junctions in Caco-2 cells

Recent studies suggest that signal transduction may have an important role in the development and regulation of the metastatic phenotype. Here, we investigated the role of the epidermal growth factor receptor (EGFR), and protein kinase C (PKC), in the process of reassembly of cadherin-dependent cell-cell adhesion of Caco-2 cells. We used chemical activation of PKC and EGFR with 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent, pretreatment with protein kinase inhibitors and subcellular fractionation to analyze the effect of the phorbol ester on the redistribution of junctional proteins. Transepithelial resistance (TER), electron microscopy and immunofluorescence analyses were also carried out. Activation with TPA resulted in disassembly of adherens junctions (AJs), but the tight junction (TJ) structure and function remained unaltered. TPA affected E-cadherin levels. In Caco-2 cells at day 2 of culture, when most E-cadherin is not associated with the cytoskeleton, a decrease in the level of this protein was observed as soon as 6 h after TPA addition. However, at day 5 of culture, the major effect observed after 6 h of treatment was a translocation of the protein from the Triton-insoluble to the -soluble fraction. On the other hand, TPA did not significantly affect the E-cadherin-associated proteins alpha and beta-catenins. Potent specific EGFR inhibitors, such as PD153035 and Tyrphostin 25, as well as Calphostin C, an inhibitor of PKC, significantly blocked the effect of TPA on AJs. Furthermore, inhibition of the TPA effect by the PD98059 MAPK inhibitor suggests that activation of this kinase was the final event in the modulation of cadherin-dependent cell-cell adhesion. Pretreatment of cell monolayers with Calphostin C before EGF treatment, one of the ligands of EGFR, blocked the redistribution of E-cadherin caused by EGF. Based on these results, we conclude that both EGFR and PKC activation are involved in TPA-induced cell signaling for modulation of cadherin-dependent cell-cell adhesion and cell shape in Caco-2 cells.     

Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells

Neurotensin (NT) and epidermal growth factor (EGF) induced rapid extracellular-regulated protein kinase (ERK) activation through different signaling pathways in the K-Ras mutated human pancreatic carcinoma cell lines PANC-1 and MIA PaCa-2. NT stimulated ERK activation via a protein kinase C (PKC)-dependent (but EGF receptor-independent) pathway in PANC-1 and MIA PaCa-2 cells, whereas EGF promoted ERK activation through a PKC-independent pathway in these cells. Concomitant stimulation of these cells with NT and EGF induced a striking increase in the duration of ERK pathway activation as compared with that obtained in cells treated with each agonist alone. Stimulation with NT + EGF promoted synergistic stimulation of DNA synthesis and anchorage-independent growth. Addition of the MEK inhibitor U0126, either prior to stimulation with NT + EGF or 2 h after stimulation with NT + EGF prevented the synergistic increase in DNA synthesis and suppressed the sustained phase of ERK activation. Furthermore, treatment with the selective PKC inhibitor GF-1 converted the sustained ERK activation in response to NT and EGF into a transient signal and also abrogated the synergistic increase in DNA synthesis. Collectively, our results suggest that the sustained phase of ERK signaling mediates the synergistic effects of NT and EGF on DNA synthesis in pancreatic cancer 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.