Recent studies indicate that maximal IL-8 protein expression requires activation of NF-κB as well as activation of the MAP kinases ERK, JNK, and p38. However, the precise relationship between NF-κB transactivation and MAP kinase activation remains unclear. We examined the requirements of NF-κB, ERK, JNK, and p38 for TNF-α-induced transcription from the IL-8 promoter in a human bronchial epithelial cell line. Treatment with TNF-α induced activation of all three MAP kinases. Using a combination of chemical and dominant-negative inhibitors, we found that inhibition of NF-κB, ERK, and JNK, but not p38, each decreased TNF-α-induced transcription from the IL-8 promoter. Inhibition of JNK signaling also substantially reduced TNF-α-induced NF-κB transactivation, whereas inhibition of ERK and p38 had no effect. On the other hand, ERK was required and sufficient for TNF-α-induced activation of activator protein (AP)-1 promoter sequences, which together function as a basal level enhancer. JNK activation was also required for AP-1 transactivation. Finally, inhibition of p38 attenuated IL-8 protein abundance, suggesting that p38 regulates IL-8 expression in a posttranscriptional manner. We conclude that, in human airway epithelial cells, MAP kinases may regulate IL-8 promoter activity by NF-κB-dependent (in the case of JNK) and -independent (ERK) processes, as well as by posttranscriptional mechanisms (p38).

Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells.

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.

Antrum mucosal protein (AMP)-18 and a synthetic peptide of amino acids 77-97 have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco-2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking whether AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Observations using laser scanning confocal (CF) microscopy supported the capacity of AMP peptide to enhance accumulation of occludin and zonula occludens-1 in TJ domains of C2 cell monolayers and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin.

An in vitro model of wound healing was used to study cell migration that is independent of proliferation during renal regeneration after acute tubular necrosis. Monolayer cultures of high-density, quiescent renal epithelial cells of the BSC-1 line were subjected to scrape wounding and then Northern blot analysis was employed to identify genes that mediate cell migration. After wounding the monolayer, there is maximal induction of the immediate-early genes Egr-1, c-fos, NAK-1, and gro at 1 hour, followed by peak induction of connective tissue growth factor (CTGF) and c-myc at 4 hours. Message levels of urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1) and heat shock protein (HSP)-70 are markedly raised 4-8 hours after wounding. Constitutive expression is repressed at 1 hour for transcripts that encode receptors for fibronectin (FN), epidermal growth factor, and hepatocyte growth factor (c-met), and the secreted proteins FN and osteopontin. Expression of genes encoding transforming growth factor (TGF)-beta 1 and -beta 2, retinoic acid receptor alpha, int-1, int-2, and gap junction protein which can play a role in cell movement, appeared unchanged after wounding. Differential expression of genes was a function of cell location relative to the wound; NAK-1, PAI-1, and HSP-70 were induced or stimulated only in cells at the wound edge, u-PA was stimulated in cells away from the wound, and CTGF was induced in each of these populations suggesting that cell-to-cell communication may regulate gene expression after wounding. Adenosine diphosphate, a potent stimulator of cell migration which enhances expression of u-PA and PAI-1 in nonwounded cultures, additively stimulates these genes after wounding and may thereby potentiate wound healing. Thus scrape wounding of renal epithelial cells is followed by induction, stimulation, or repression of specific genes with distinct responses in different populations of cells.

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D(1) promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D(1) promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110(PI) (3-K)CAAX) was sufficient to activate the cyclin D(1) promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110(PI) (3-K)CAAX was insufficient to activate ERK. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was not blocked by PD98059, an inhibitor of mitogen-activated protein kinase/ERK kinase. We next examined whether PI 3-kinase and the 21-kD guanidine triphosphatase Rac1 regulate cyclin D(1) promoter activity by similar mechanisms. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was decreased by two inhibitors of Rac1-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rac1 each activated the cyclin D(1) promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D(1) promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D(1) promoter.

When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor alpha 2 cDNA at low stringency, we found a weakly hybridizing cDNA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (Ko.5 0.3 ng/ml) stimulated migration, whereas transforming growth factor-,82 was inhibitory (K; -0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function. (J. Clin. Invest. 1992. 90:288-292.) Key words: acute renal failure * transforming growth factor-,@ * epidermal growth factor * heparin . extracellular matrix