Bi-directional regulation of Ser-985 phosphorylation of c-met via protein kinase C and protein phosphatase 2A involves c-Met activation and cellular responsiveness to hepatocyte growth factor

Previous studies indicated that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate induced phosphorylation of Ser-985 at the juxtamembrane of c-Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), and this was associated with decreased tyrosine phosphorylation of c-Met. However, the regulatory mechanisms and the biological significance of the Ser-985 phosphorylation in c-Met remain unknown. When A549 human lung cancer cells were exposed to oxidative stress with H(2)O(2), H(2)O(2) treatment induced phosphorylation of Ser-985, but this was abrogated by an inhibitor for protein kinase C (PKC). Likewise, treatment of cells with NaF (an inhibitor of protein phosphatases) allowed for phosphorylation of Ser-985, and a protein phosphatase responsible for dephosphorylation of Ser-985 was identified to be protein phosphatase 2A (PP2A). The effects of PKC inhibitors revealed that PKCdelta and -epsilon were responsible for the Ser-985 phosphorylation of c-Met, and pull-down analysis indicated that associations of PKCdelta and -epsilon with c-Met may be involved in the regulation of Ser-985 phosphorylation of c-Met. Instead, PP2A was constitutively associated with c-Met, whereas its activity to dephosphorylate Ser-985 of c-Met was decreased when cells were exposed to H(2)O(2). Addition of HGF to A549 cells in culture induced c-Met tyrosine phosphorylation, the result being mitogenic response and cell scattering. In contrast, in the presence of H(2)O(2) stress, HGF-dependent tyrosine phosphorylation of c-Met was largely suppressed with a reciprocal relationship to Ser-985 phosphorylation, and this event was associated with abrogation of cellular responsiveness to HGF. These results indicate that Ser-985 phosphorylation of c-Met is bi-directionally regulated through PKC and PP2A, and the Ser-985 phosphorylation status may provide a unique mechanism that confers cellular responsiveness/unresponsivenss to HGF, depending on extracellular conditions.     

Does heme oxygenase-1 have a role in Caco-2 cell cycle progression?

Intestinal epithelium undergoes a rapid self-renewal process characterized by the proliferation of the crypt cells, their differentiation into mature enterocytes as they migrate up to the villi, followed by their shedding as they become senescent villus enterocytes. The exact mechanism that regulates the intestinal epithelium renewal process is not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role. Heme oxygenase-1 (HO-1) is involved in endothelial cell cycle progression, but its role in the intestinal epithelial cell turnover has not been explored. With its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process. In this study, we examined the role of HO-1 in the proliferation and differentiation of Caco-2 cells, a well-established in vitro model for human enterocytes. After confluence, Caco-2 cells undergo spontaneous differentiation and mimic the crypt to villus maturation observed in vivo. In preconfluent and confluent Caco-2 cells, HO-1 protein expression was determined with the immunoblot. HO-1 activity was determined by the ability of the enzyme to generate bilirubin from hemin. The effect of a HO-1 enzyme activity inhibitor, tin protoporphyrin (SnPP), on Caco-2 cell proliferation and differentiation was examined. In preconfluent cells, cell number was determined periodically as a marker of proliferation. Cell viability was measured with MTT assay. Cell differentiation was assessed by the expression of a brush border enzyme, alkaline phophatase (ALP). HO-1 was expressed in subconfluent Caco-2 cells and remained detectable until 2 days postconfluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the ALP enzyme activity of confluent cells. HO-1 may be involved in intestinal cell cycle progression.     

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.     

Villin enhances hepatocyte growth factor-induced actin cytoskeleton remodeling in epithelial cells

Villin is an actin-binding protein localized to intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a calcium-dependent manner. Although villin is not necessary for the bundling of F-actin in vivo, it is important for the reorganization of the actin cytoskeleton elicited by stress during both physiological and pathological conditions (Ferrary et al., 1999). These data suggest that villin may be involved in actin cytoskeleton remodeling necessary for many processes requiring cellular plasticity. Here, we study the role of villin in hepatocyte growth factor (HGF)-induced epithelial cell motility and morphogenesis. For this purpose, we used primary cultures of enterocytes derived from wild-type and villin knock-out mice and Madin-Darby canine kidney cells, expressing villin in an inducible manner. In vitro, we show that epithelial cell lysates from villin-expressing cells induced dramatic, calcium-dependent severing of actin filaments. In cell culture, we found that villin-expressing cells exhibit enhanced cell motility and morphogenesis upon HGF stimulation. In addition, we show that the ability of villin to potentiate HGF-induced actin reorganization occurs through the HGF-activated phospholipase Cgamma signaling pathway. Collectively, these data demonstrate that villin acts as a regulator of HGF-induced actin dynamics.     

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by −20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and ³H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation. © 1996 Wiley-Liss, Inc.     

Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer

The human intestinal Caco-2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new cell-maintenance protocol in which Caco-2 cells were subcultured at 50% of confluence instead of 80% of confluence, as usually suggested. Using this new protocol, Caco-2 cells retained a higher proliferation potential resulting in a cell population, which, on reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21-days differentiated monolayers by microarrays hybridization and by confocal microscopy. We then investigated if these differences could also modify the effects of toxicants on 21-days-differentiated cells. We analyzed the 2 h-acute toxicity of CuCl(2) in terms of actin depolymerization and metallothionein 2A (MT2A) and heat shock protein 70 (HSPA1A) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with culture protocol, the low-density growing cells showing a more homogeneous and stronger response. Our results suggest that cell growing density could influence a number of morphological and physiological properties of differentiated Caco-2 cells and these effects must be taken in account when these cells are used as intestinal model.     

Activation of HGF/c-Met signaling by ultrafine carbon particles and its contribution to alveolar type II cell proliferation

Hepatocyte growth factor (HGF) is a potent mitogen and motogen for various epithelial cells. The present study aimed to explore the role of HGF and c-Met receptor in ultrafine carbon particle-induced alveolar type II epithelial (type II) cell proliferation. ICR mice were intratracheally instilled with 100 μg ultrafine carbon black (ufCB) and killed at 21, 48, and 72 days postexposure to examine type II cell proliferation, HGF release, and c-Met activation. In vivo and in vitro applications of neutralizing anti-HGF antibody were used to investigate the causal role of HGF in cell proliferation. The Met kinase inhibitor SU11274 and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 were used to delineate the involvement of c-Met/ERK1/2 in rat L2 pulmonary epithelial cell proliferation. The results demonstrated that in vivo exposure to 100 μg ufCB caused increased HGF in bronchoalveolar lavage fluid, as well as increased HGF production, c-Met phosphorylation, and cell proliferation in type II cells. In vitro study revealed that ufCB caused a dose-dependent increase in HGF release, c-Met phosphorylation, and cell proliferation. Importantly, treatment with the neutralizing anti-HGF antibody significantly blocked ufCB-induced in vivo and in vitro type II cell proliferation. Moreover, SU11274 and PD98059 significantly reduced ufCB-increased L2 cell proliferation. Results from Western blotting demonstrated that SU11274 successfully suppressed ufCB-induced phosphorylation of c-Met and ERK1/2. In summary, the activation of HGF/c-Met signaling is a major pathway involved in ufCB-induced type II cell proliferation.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Cyclic AMP-dependent protein kinase A negatively modulates adherens junction integrity and differentiation of intestinal epithelial cells

Intestinal epithelial cell differentiation is a complex process in which many different signaling pathways are likely involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to inhibit enterocyte differentiation; however, the mechanisms through which cAMP/PKA signaling modulates differentiation of human intestinal epithelial cells are still not well understood. Herein, we report that: (1) treatment of Caco-2/15 cells with 8Br-cAMP repressed sucrase-isomaltase and villin protein expression and strongly attenuated morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush border formation; (2) treatment of confluent Caco-2/15 cells with 8Br-cAMP led to a strong decrease in F-actin localized at cell-cell contact sites along with a reduced amount of E-cadherin and catenins, but not of ZO-1, at cell-cell interfaces concomitant with a decreased association of these proteins with the actin cytoskeleton; (3) inhibition of PKA by H89 prevented disruption of adherens junctions by extracellular calcium depletion; (4) treatment of Caco-2/15 cells with 8Br-cAMP prevented the recruitment and activation of p85/PI-3K to E-cadherin-mediated cell-cell contacts, an important event in the assembly of adherens junctions and differentiation of these cells; (5) E-cadherin appears to be phosphorylated on serine in vivo in a PKA-dependent mechanism. Conclusion: Our studies show that cAMP/PKA signaling negatively regulates adherens junction integrity as well as morphological and functional differentiation of intestinal epithelial cells.     

Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells

Gene expression of activin, activin receptors, and follistatin was investigated in vivo and in vitro using semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and the intestinal epithelial cell line IEC-6 expressed mRNA encoding the betaA-subunit of activin, alpha-subunit of inhibin, activin receptors IB and IIA, and follistatin. An epithelial cell isolation study focused along the crypt-villus axis in rat jejunum showed that betaA mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a differentiation model of enterocytes. Four- to fivefold induction of betaA mRNA was observed in postconfluent Caco-2 cells grown on filter but not in those cells grown on plastic. In contrast, follistatin mRNA was seen to be reduced after reaching confluence. Exogenous activin A dose-dependently suppressed the proliferation and stimulated the expression of apolipoprotein A-IV gene, a differentiation marker, in IEC-6 cells. These results suggest that the activin system is involved in the regulation of such cellular functions as proliferation and differentiation in intestinal epithelial cells.     

PKC mediates fluctuant ERK-paxillin signaling for hepatocyte growth factor-induced migration of hepatoma cell HepG2

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24 h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24 h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.     

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.     

PKC controls HGF-dependent c-Met traffic, signalling and cell migration

The growth factor/receptor pair HGF/c-Met exerts control on proliferation, morphogenesis and motility, and through overexpression and mutation is implicated in cancer. Here we have investigated the relationship between receptor signalling and traffic, and its control by specific PKC isotypes. It is shown that c-Met signalling to the ERK cascade occurs within endosomal compartments and that it is in this compartment that PKCepsilon specifically exerts its control on the pathway with the consequent accumulation of ERK in focal complexes. These events are clearly separated from the subsequent microtubule-dependent sorting of c-Met to its perinuclear destination, which is shown to be under the control of PKCalpha. Thus while it is shown that traffic to endosomes is essential for HGF/c-Met to trigger an ERK response, the subsequent traffic and signalling of c-Met controlled by these two PKC isotypes are unconnected events. The dynamic properties conferred by the PKCepsilon control are shown to be essential for a normal HGF-dependent migratory response. Thus PKCs are shown to control both receptor traffic and signal traffic to relay HGF/c-Met responses.     

Molecular imaging of c-Met tyrosine kinase activity

The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities and can lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/c-Met receptor coupling or c-Met signaling events in real time and in living cells and animals, here we describe a genetically engineered reporter where bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. c-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor-bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter’s bioluminescence activity in a dose-dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.     

Modulation of human Caco-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10^-12M-10^-4 M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.