Inhibitory effect of taxol, a microtubule stabilizing agent, on induction of DNA synthesis is dependent upon cell lines and growth factors.

Growth-arrested rat fibroblasts, 3Y1, and human diploid fibroblasts, TIG-1, were induced to synthesize DNA by stimulation with various agents such as fetal bovine serum (FBS), epidermal growth factor (EGF), colcemid, or colchicine. Taxol, a microtubule-stabilizing agent, blocked the induction of DNA synthesis after stimulation with colcemid or colchicine in both cell lines. Taxol inhibited the induction of DNA synthesis after stimulation with FBS or EGF in TIG-1, but did not in 3Y1. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in TIG-1, which was reduced only partly by taxol. Taxol stabilized or polymerized microtubules in both cell lines. These results indicate that the inhibitory effect of taxol on the induction of DNA synthesis varied among cell lines and among growth factors, and suggest that signal transduction processes may be differentiated by taxol sensitivity. In TIG-1 cells, when taxol was added within 6 h, about halfway into the initiation of DNA synthesis after the addition of FBS or EGF, the inhibition of DNA synthesis still occurred. Taxol did not inhibit the induction of c-fos and c-myc genes by FBS or EGF stimulation. Colchicine itself did not induce these genes in TIG-1. Thus, taxol appeared to inhibit the induction of DNA synthesis not by blockage in the early transduction process of the growth signal from the cell surface to nuclei but by blockage in processes operating in the mid- or late-prereplicative phase.     

The absence of prostaglandin e1 returned confluent cultures of highly proliferative murine polycystic kidney principal cells to a normal proliferation level

Constitutively high proliferation, loss of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA)-regulated proliferation, and half-normal cAMP levels were observed previously in principal cells from the C57BL/6J- Cyc1\[cf12\]cpk\[cf1\] (cpk) model of autosomal recessive polycystic kidneys disease (PKD) cultured in defined medium supplemented with prostaglandin E1 (PGE1). Because PGE1 can up- or down-regulate renal cAMP production depending upon its receptor coupling; cAMP exerted both PKA-dependent and PKA-independent effects on cell proliferation; proliferation is considered to be a component of cystogenesis; and PGE1 resulted in loss of tubular structures and formation of cystic structures in gel culture of Madin Darby Canine Kidney cells; the effect of removing PGE1 on murine principal cell proliferation was examined. Proliferation was measured in filter-grown cultures of cystic (cpk) and noncystic (C57) principal cells from cpk and C57BL/6J mice, respectively. Lack of PGE1 had no effect on subconfluent C57 and cpk cultures or confluent C57 cultures but had a dramatic effect on confluent cpk cultures. Without PGE1, cpk proliferation was comparable with the low C57 level. In PGE1-deficient medium, differences were observed between confluence conditions and cell types for responses to a cAMP analog and a PKA activity inhibitor that suggested altered regulation of both PKA-dependent and PKA-independent cell proliferation. Cyclic adenosine monophosphate-dependent differences reported here, and previously, support the idea that the combination of mutant PKD gene product, altered PGE1 responsiveness, and altered PKA targeting contributes to activation of a cystogenic signaling pathway that regulates principal cell proliferation and is involved in pathogenesis.     

cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia

Developmental control of cellproliferation is crucial, and abnormal principal cell proliferation maycontribute to cystogenesis in polycystic kidney disease. This studyinvestigates roles of cAMP and its primary effector, cAMP-dependentprotein kinase (protein kinase A; PKA), in control of cellproliferation in filter-grown noncystic (NC) and cystic (CY)-derivedprincipal cell cultures. These cultures had similar cAMP pathwaycharacteristics upstream of PKA subunit distribution but differed inpredicted PKA subtype distribution. Functionally, cultures wereproliferative before polarization, with constitutively higherproliferation in CY cultures. NC cultures achieved levels similar tothose of CY cultures on pharmacological manipulation of cAMP productionor PKA activation or inhibition of PKA subtype I activity. Inhibitionof overall PKA activity, or of PKA subtype II anchoring, diminishedcAMP/PKA-mediated proliferation in NC cultures but had no effect on CYcultures. Polarized CY monolayers remained proliferative, but NCmonolayers lost responsiveness. No large proliferation changes resultedfrom treatments of polarized cultures; however, polarized NC and CY cultures differed in poststimulation handling of PKA catalytic and typeII regulatory subunits. Our results support PKA subtype regulationof prepolarization proliferation in NC principal cells and alteredregulation of PKA in CY cells and suggest that differences at ordownstream of PKA can contribute to altered proliferation in adevelopmental renal disease.

     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Differential stimulation of growth related metabolism in cultured smooth muscle cells from SHR and WKY rats by combinations of EGF and LDL

We have reported previously that vascular smooth muscle cells from spontaneously hypertensive rats (SHR) were more responsive to epidermal growth factor (EGF) than their normotensive derived Wistar Kyoto (WKY) controls. This differential responsiveness is evident for several cellular processes including activation of S6-kinase, elevation of intracellular pH and stimulation of both phosphoinositide metabolism and DNA synthesis. Quiescent smooth muscle cells exposed to low density lipoprotein (LDL) exhibited a similar differential responsiveness (SHR greater than WKY) in terms of S6-kinase activation, which was time- and dose-dependent (10(-10)-10(7) M), but neither cell type responded appreciably to LDL in terms of a stimulation in [3H]-thymidine incorporation. Exposure of the same cells to EGF and LDL in combination elicited a marked synergistic stimulation in DNA synthesis, the extent of which was greater for SHR than WKY. The sensitivity of both cell types to EGF was increased in the presence of LDL, although cells from hypertensive animals still exhibited their greater (vs. WKY) sensitivity. In both cell types, activation of nuclear protooncogenes c-fos and c-myc by LDL was minimal, whereas oncogene induction by EGF was approximately five-fold greater for SHR-derived cells compared to those from WKY animals. No marked synergistic effect on the time-dependent induction of either entity was observed for cells exposed to EGF and LDL simultaneously, and the response of SHR-cells remained greater than WKY-cells.     

Regulation of c-fos expression in primary culture of guinea pig glandular epithelial cells stimulated by growth factors and estradiol

The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.     

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.     

Bypass of the ts block of tsJT60, a G0-specific ts mutant from rat fibroblasts, by fetal bovine serum and epidermal growth factor

tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.     

Growth factor-mediated K+ channel activity associated with human myeloblastic ML-1 cell proliferation

ML-1 cell proliferation is dependent on the presence of serumgrowth factors. Removing serum from the culture medium results ingrowth arrest and promotes differentiation. In this study, we foundthat a 4-aminopyridine-sensitiveK⁺ channel was highly expressed inproliferating ML-1 cells and significantly diminished inG₁-arrested ML-1 cells induced by serum deprivation but was restored within 30 min in these cells withaddition of 10% fetal bovine serum (FBS) or 5 ng/ml epidermal growthfactor (EGF). Intracellular adenosine 3',5'-cyclicmonophosphate (cAMP) levels, but not guanosine 3',5'-cyclicmonophosphate, were significantly increased in serum-deprived cellsstimulated by FBS or EGF, and the effects of FBS and EGF on the channelactivation were mimicked by exogenous cAMP. In inside-out patches,K⁺ channel activity wassignificantly increased by the cAMP-dependent protein kinase catalyticsubunit, whereas the effect of EGF on K⁺ channel activation was blockedby Rp-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphothioate. Together, our resultsdemonstrate that serum growth factors stimulateK⁺ channel activity inproliferation of ML-1 cells through protein kinase-inducedphosphorylation and suggest an important molecular mechanism for serumgrowth factor-stimulated mitogenesis in ML-1 cells.

     

Early stimulation by EGF plus insulin of rRNA, c-fos, and actin mRNA expression: Inhibition by cytochalasin D

Stimulation of membrane ruffling is one of the first events induced by addition of growth factors to quiescent cultures. In order to assess the importance of intact cytoskeleton in induction, by EGF + insulin, of early events such as stimulation of rRNA, c-fos, and actin mRNA expression, we studied the effect of cytochalasin D (CD) on these metabolisms. We observed that CD slightly increased rRNA synthesis in nonstimulated cells; conversely, it decreased rRNA synthesis in cells stimulated by EGF + insulin. The maximal inhibition observed was 60%. The c-fos mRNA was not expressed in control cells and was accumulated in cells stimulated by the mixture of EGF + insulin; this accumulation was inhibited by CD. Actin mRNA was expressed in control cells and its expression was stimulated by EGF + insulin. Addition of CD decreased actin mRNA accumulation in stimulated cells but increased this accumulation in unstimulated cells. Our results, taken together, show that CD specifically affected the stimulation of rRNA and mRNA expression induced by growth factors and suggest that intact cytoskeleton and possibly membrane ruffling favored this stimulation.     

The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones

Epidermal growth factor (EGF) added in a single dose (between 10^–16 and 1.7 ± 10^–9M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (³H)thymidine labeling and autoradiography and by a 1-hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24-hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum-devoid medium EGF (10^–11M) enhanced hepatocytic mitotic, but not DNA-synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non-parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10^–14M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA-synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24-hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000-fold higher EGF concentration, and was twice as active as either an equimolar (10^–14M) mixture of the two pancreatic hormones or EGF by itself at 10^–14M. These results show that the growth-promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.     

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.     

Transforming growth factor alpha (TGF alpha) induction of C-FOS and C-MYC expression in C3H 10T1/2 cells

We have investigated the effects of transforming growth factor alpha (TGF alpha) in C3H10T1/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGF alpha, which both utilize the EGF receptor for signal generation, are able to stimulate DNA synthesis in these cells with nearly superimposable kinetics; however, the stimulation by TGF alpha was slightly greater at nearly all time points assayed. This report is the first showing that TGF alpha, like EGF, vigorously induces c-myc and c-fos gene expression in these cells. A significant stimulation of c-myc and c-fos mRNA levels is observed with both TGF alpha and EGF; c-myc mRNA levels show an 8-fold induction with both mitogens, while c-fos inductions were on the order of 12 to 14-fold at maximum. However, the induction of c-myc mRNA by TGF alpha has slower kinetics than by EGF.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Cyclic AMP-mediated cyst expansion

In polycystic kidney disease (PKD), intracellular cAMP promotes cyst enlargement by stimulating mural epithelial cell proliferation and transepithelial fluid secretion. The proliferative effect of cAMP in PKD is unique in that cAMP is anti-mitogenic in normal renal epithelial cells. This phenotypic difference in the proliferative response to cAMP appears to involve cross-talk between cAMP and Ca²⁺ signaling to B-Raf, a kinase upstream of the MEK/ERK pathway. In normal cells, B-Raf is repressed by Akt (protein kinase B), a Ca²⁺-dependent kinase, preventing cAMP activation of ERK and cell proliferation. In PKD cells, disruption of intracellular Ca²⁺ homeostasis due to mutations in the PKD genes relieves Akt inhibition of B-Raf, allowing cAMP stimulation of B-Raf, ERK and cell proliferation. Fluid secretion by cystic cells is driven by cAMP-dependent transepithelial Cl⁻ secretion involving apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels. This review summarizes the current knowledge of cAMP-dependent cyst expansion, focusing on cell proliferation and Cl⁻-dependent fluid secretion, and discusses potential therapeutic approaches to inhibit renal cAMP production and its downstream effects on cyst enlargement. This article is part of a Special Issue entitled: Polycystic Kidney Disease.