Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.

Urokinase-type plasminogen activator (uPA) is an extracellular protease and expressed in various cells that exhibit dynamic changes in cell morphology, suggesting a link between cytoskeletal reorganization (CSR) and uPA expression. CSR can be induced by pharmacological agents, such as by colchicine for microtubule cytoskeleton and by cytochalasin for microfilament cytoskeleton. Using these agents, we previously showed that CSR induced the uPA gene in LLC-PK1 cells independently of the protein kinase C and cAMP-dependent protein kinase. Here we show that the induction of the uPA gene by CSR is mediated by the activation of c-Jun which interacts with an AP-1-like site located 2 kb upstream of the uPA gene. 12-O-tetradecanoylphorbol 13-acetate (TPA) induces the uPA gene through the same elements, but additionally utilizes an adjacent PEA3 element and induces c-fos. Furthermore, CSR induces a greater accumulation and a more pronounced phosphorylation of c-Jun than TPA induction. AP-1 is a positive regulator of growth and oncogenesis, and CSR is an integral part of these processes. Our results provide a view how CSR and AP-1 could be coupled in these processes. We also show that TPA and CSR act synergistically, suggesting a model where an initial activation signal could be amplified by CSR.     

Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.     

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition.

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.     

Stimulation of plasminogen activator expression and induction of DNA synthesis by microtubule-disruptive drugs

Treatment of confluent Swiss 3T3 cells in serum-free medium with colchicine, a drug known to depolymerize microtubules, results in a dose-dependent increase in both released and cell-associated plasminogen activator levels. Other anti-microtubule drugs (vinblastine and nocodazole) are also active in stimulating plasminogen activator expression. In contrast, cytochalasin B, a microfilament-disruptive drug, has no effect. In addition, treatment with colchicine, vinblastine or nocodazole, but not cytochalasin B, also results in a dose-dependent induction of DNA synthesis in both confluent and quiescent sparse 3T3 cells in the absence of serum. Furthermore, colchicine treatment also mediates a marked morphologic change. Thus, disruption of microtubules may be sufficient to render 3T3 cells in an “activated” state characterized by morphologic alteration, enhanced plasminogen activator expression and induction of DNA synthesis.     

Role of the cytoskeleton in laminin induced mammary gene expression

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.     

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.     

Drug-induced alterations in rat peritubular cell cytoskeleton result in proteoglycan synthesis modifications. Comparison with some intracellular signaling pathways.

The influence of phorbol myristate acetate (PMA), dibutyryl cAMP and insulin-like growth factor (IGF-1) as well as cytoskeletal disrupting drugs on morphological changes has been studied in peritubular cells isolated from immature rat testis. Morphological studies were combined with immunofluorescence investigations of cytoskeletal elements and their rearrangements by various agents. The results were correlated with modulation of proteoglycan synthesis. Peritubular cells exposed to dibutyryl cAMP or cytochalasin D were transformed from flattened, fibroblast-like into neuronal-like morphology. In such cells, destruction of actin filaments was accompanied with a 50% decrease in cell-associated proteoglycan synthesis as well as with oversulfation of total proteoglycans. On the contrary, peritubular cell shape has been slightly altered after addition of PMA, IGF-1, vinblastine or colchicine. After these treatments, destruction or rearrangement of cytoskeletal elements was observed; cell-layer proteoglycan synthesis remained either unchanged or increased while total proteoglycans were always undersulfated. IGF-1, PMA and dibutyryl cAMP modified the peritubular cell morphology, cytoskeletal organization and proteoglycan production; the cytoskeleton disrupting drugs such as vinblastine, colchicine and cytochalasin D mimicked some of these effects. These observations suggest that alterations in proteoglycan biosynthesis, after activation of tyrosine kinase, protein kinase C and protein kinase A pathways might be mediated, at least in part, by the disorganization of the cytoskeleton structure.     

Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton

Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction of CTGF by transforming growth factor beta (TGF-beta) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediated CTGF induction was due to activation of 5-HT(2A) receptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-beta. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction of CTGF by LPA and TGF-beta. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulated CTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGF expression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants of CTGF expression induced by LPA and 5-HT, and also by TGF-beta.     

Cytoskeletal reorganization leads to induction of the urokinase-type plasminogen activator gene by activating FAK and Src and subsequently the Ras/Erk signaling pathway.

Previously, we showed that cytoskeletal reorganization (CSR) induced by colchicine or cyochalasins leads to activation of the urokinase-type plasminogen activator (uPA) gene in LLC-PK(1) cells via the Ras/Erk signaling pathway [Irigoyen et al. (1997) J. Biol. Chem. 272, 1904]. It remained to be seen how CSR activates Ras/Erk signaling. Changes in cell morphology triggered by extracellular signals are often mediated by integrin-associated proteins, such as focal adhesion kinase (FAK) and Src. We found that CSR induced the activation of FAK and Src and the association of FAK and Shc, a signaling molecule linking growth factor receptor tyrosine kinase and Grb2. Furthermore, expression of either FRNK, a kinase-minus FAK-like molecule acting as a dominant negative FAK, or a dominant negative Src suppressed CSR-induced uPA gene promoter activation. These results suggest that cells respond to a morphology change, using the cytoskeleton as a sensor, by activating FAK and Src and subsequently the Ras/Erk signaling pathway.     

Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter.

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.     

Cytochalasin D enhances the accumulation of a protease‐resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway

The conversion of a host‐encoded PrPsen (protease‐sensitive cellular prion protein) into a PrPres (protease‐resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion‐infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule‐disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3‐kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D‐induced enhancement of PrPres accumulation. Cytochalasin D‐induced extension of neurite‐like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion‐infected cells.     

The relationship between the disassembly of microtubules during G1 and the enhancement of DNA synthesis by colchicine in mouse fibroblasts stimulated with peptide growth hormones

By immunofluorescent staining to visualize the cytoplasmic microtubular cytoskeleton in mouse fibroblasts we have ascertained that after a relatively short exposure of cells to colchicine, microtubules remain disassembled for a prolonged period of time after cells are transferred to a colchicine-free medium. In contrast to the persisting effects of colchicine, a brief exposure of cells to nocodazole first induces the expected disruption of microtubules followed by regeneration of the cytoskeleton within a few hours after removal of extracellular drug. These results shed light on our previous finding that quiescent mouse fibroblasts first treated with colchicine and then transferred to colchicine-free medium exhibit an enhanced proliferative response to EGF and insulin, whereas cells treated in a similar manner with nocodazole show no enhancement of DNA synthesis stimulated by peptide growth hormones. We conclude that cytoplasmic microtubules must remain disaggregated during the prereplicative G1 period in order for cells to exhibit the enhancing effects of the microtubule-disrupting drugs on DNA synthesis.     

Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.