Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.     

Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells

Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 microg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH(2)-terminal kinase, Akt/protein kinase B, and p90(RSK). FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [(3)H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.     

Hepatocyte growth factor/scatter factor stimulates migration of rat mammary fibroblasts through both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways.

Hepatocyte growth factor/scatter factor (HGF/SF) is considered to be a mesenchymal-derived factor that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 fibroblasts, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-15 min after the addition of the growth factor, which was followed by a rapid decrease to near basal levels after 20 min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated 15 min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of fibroblasts. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 fibroblasts and that these pathways are mutually dependent.     

Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

Prostaglandin F2alpha involves heparan sulphate sugar chains and FGFRs to modulate osteoblast growth and differentiation

The present investigation extends our previous studies on PGF2alpha-mediated signalling in osteoblast metabolism. In particular, the role of PGF2alpha as modulator of heparan sulphate proteoglycans (HSPGs), fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptors (FGFRs) was evaluated. We hereby reported the novel observation that PGF2alpha was able to promote the formation of HSPGs/FGF-2/FGFRs complexes. Moreover, our data suggested that PGF2alpha could induce new synthesis of heparan sulphate (HS) chains on osteoblasts by a mechanism involving a modulation of MAPK signalling and that HS is required for the regulation of FGF-2 induced by PGF2alpha. Indeed, a proteolytic cleavage of HSPGs with heparinase III (Hep III) prior to PGF2alpha administration down-regulated the basal expression of phospho-p44/42, likely inhibiting FGFRs tyrosine kinase activity. Interestingly, MAPK signalling influenced syntheses and subcellular localization of FGF-2, its specific receptor and HS. In addition, the proteolytic cleavage by Hep III and the MAPK kinase inhibition by PD-98059 also revealed that PGF2alpha induced cell proliferation is dependent on HSPGs and FGF-2 specific receptor, respectively. Of further relevance of this study, we demonstrated, by using a specific siRNA for FGFR1, that PGF2alpha modulates Runx2 expression by FGFR1 and HS.     

Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.     

Regulation of constitutive p50/c-Rel activity via proteasome inhibitor-resistant IkappaBalpha degradation in B cells

Constitutive NF-kappaB activity has emerged as an important cell survival component of physiological and pathological processes, including B-cell development. In B cells, constitutive NF-kappaB activity includes p50/c-Rel and p52/RelB heterodimers, both of which are critical for proper B-cell development. We previously reported that WEHI-231 B cells maintain constitutive p50/c-Rel activity via selective degradation of IkappaBalpha that is mediated by a proteasome inhibitor-resistant, now termed PIR, pathway. Here, we examined the mechanisms of PIR degradation by comparing it to the canonical pathway that involves IkappaB kinase-dependent phosphorylation and beta-TrCP-dependent ubiquitylation of the N-terminal signal response domain of IkappaBalpha. We found a distinct consensus sequence within this domain of IkappaBalpha for PIR degradation. Chimeric analyses of IkappaBalpha and IkappaBbeta further revealed that the ankyrin repeats of IkappaBalpha, but not IkappaBbeta, contained information necessary for PIR degradation, thereby explaining IkappaBalpha selectivity for the PIR pathway. Moreover, we found that PIR degradation of IkappaBalpha and constitutive p50/c-Rel activity in primary murine B cells were maintained in a manner different from B-cell-activating-factor-dependent p52/RelB regulation. Thus, our findings suggest that nonconventional PIR degradation of IkappaBalpha may play a physiological role in the development of B cells in vivo.     

Basic fibroblast growth factor inhibits radiation-induced apoptosis of HUVECs. II. The RAS/MAPK pathway and phosphorylation of BAD at serine 112

Radiation-induced endothelial cell apoptosis is involved in the development of many radiation injuries, including radiation-induced skin ulcers. The proangiogenic growth factor basic fibroblast growth factor (bFGF, NUDT6) enhances endothelial cell survival. In the present study, we set up a model of apoptosis in which primary cultured human umbilical vein endothelial cells (HUVECs) were irradiated with (60)Co gamma rays to explore the effects of bFGF on radiation-induced apoptosis of HUVECs and the signaling pathways involved. We found that bFGF inhibited radiation-induced apoptosis of HUVECs, and that the effect was mediated in part by the RAS/MEK/ MAPK/RSK (p90 ribosomal S6 kinase)/BAD pathway. This pathway was activated by exposure of irradiated HUVECs to bFGF, involving phosphorylation of FGFR, MEK and p44/42 MAPK. The survival-enhancing effect of bFGF was partly inhibited by U0126 and PD98059. The fact that the anti-apoptosis effect of bFGF on irradiated HUVECs was not completely abrogated by U0126 and PD98059 suggests that other survival signaling pathways may exist. Transfection of a dominant-negative form of RSK2 (DN RSK2) partly blocked the anti-apoptosis effect of bFGF in irradiated HUVECs. Moreover, we provide evidence for the first time that bFGF induced BAD phosphorylation (at serine 112) and CREB (cAMP response element-binding protein) activation (phosphorylation at serine 133) in gamma-irradiated HUVECs. In our model, inhibition of MAPK signaling-dependent phosphorylation of BAD at serine 112 promoted increased association with BCL-X(L), suggesting that MAPK pathway-dependent serine 112 phosphorylation of BAD is critical for the effect of bFGF on cell survival. These results showed that RAS/MAPK/BAD pathway participated in the bFGF-induced effect on survival of HUVECs exposed to radiation. It is suggested that RAS/ MAPK pathway in tumor vascular endothelium could be a potential therapeutic target to enhance the efficacy of ionizing radiation.     

AMP-activated protein kinase positively regulates FGF-2-stimulated VEGF synthesis in osteoblasts

AMP-activated protein kinase (AMPK) is recognized as a regulator of energy homeostasis. We have previously reported that basic fibroblast growth factor (FGF-2) stimulates vascular endothelial growth factor (VEGF) release through the activation of p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMPK in FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an AMPK inhibitor, which suppressed the FGF-2-induced phosphorylation of AMPK, significantly inhibited the VEGF release stimulated by FGF-2. The AMPK inhibitor also reduced the mRNA expression of VEGF induced by FGF-2. The FGF-2-induced phosphorylation of both p44/p42 MAP kinase and SAPK/JNK was attenuated by compound C. These results strongly suggest that AMPK positively regulates the FGF-2-stimulated VEGF synthesis via p44/p42 MAP kinase and SAPK/JNK in osteoblasts.     

TNF activates Syk protein tyrosine kinase leading to TNF-induced MAPK activation, NF-kappaB activation, and apoptosis

Spleen tyrosine kinase (Syk), a nonreceptor protein kinase initially found to be expressed only in hemopoietic cells, has now been shown to be expressed in nonhemopoietic cells and to mediate signaling of various cytokines. Whether Syk plays any role in TNF signaling was investigated. Treatment of Jurkat T cells with TNF activated Syk kinase but not ZAP70, another member of Syk kinase family, and the optimum activation occurred at 10 s and with 1 nM TNF. TNF also activated Syk in myeloid and epithelial cells. TNF-induced Syk activation was abolished by piceatannol (Syk-selective inhibitor), which led to the suppression of TNF-induced activation of c- JNK, p38 MAPK, and p44/p42 MAPK. Jurkat cells that did not express Syk (JCaM1, JCaM1/lck) showed lack of TNF-induced Syk, JNK, p38 MAPK, and p44/p42 MAPK activation, as well as TNF-induced IkappaBalpha phosphorylation, IkappaBalpha degradation, and NF-kappaB activation. TNF-induced NF-kappaB activation was enhanced by overexpression of Syk by Syk-cDNA and suppressed when Syk expression was down-regulated by expression of Syk-small interfering RNA (siRNA-Syk). The apoptotic effects of TNF were reduced by up-regulation of NF-kappaB by Syk-cDNA, and enhanced by down-regulation of NF-kappaB by siRNA-Syk. Immunoprecipitation of cells with Syk Abs showed TNF-dependent association of Syk with both TNFR1 and TNFR2; this association was enhanced by up-regulation of Syk expression with Syk-cDNA and suppressed by down-regulation of Syk using siRNA-Syk. Overall, our results demonstrate that Syk activation plays an essential role in TNF-induced activation of JNK, p38 MAPK, p44/p42 MAPK, NF-kappaB, and apoptosis.     

Ikappakappa mediates NF-kappaB activation in human immunodeficiency virus-infected cells

Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-kappaB in the nucleus secondary to increased IkappaBalpha, IkappaBbeta, and IkappaBepsilon degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IkappaB, we have sought to identify the regulatory domains of IkappaBalpha targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IkappaBalpha, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IkappaBalpha. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IkappaBalpha degradation and NF-kappaB activation. Of the putative N-terminal IkappaBalpha kinases, we demonstrated that the Ikappakappa complex, but not p90(rsk), is activated by HIV infection and mediates HIV-dependent NF-kappaB activation. Analysis of viral replication in cells that constitutively express IkappaBalpha negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-kappaB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.     

Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome

Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IkappaBalpha and IkappaBbeta and the related protein NF-kappaB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-kappaB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-kappaB was still observed. Cysteine peptidase inhibitors prevented IkappaBalpha, IkappaBbeta, and NF-kappaB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IkappaBalpha in vitro. LPS-mediated IkappaBalpha and IkappaBbeta degradation was not affected by these inhibitors, confirming that the site of degradation of IkappaBalpha, IkappaBbeta, and NF-kappaB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-kappaB and consequently inhibit IL-12 production.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Up-regulation by zinc of FGF-2-induced VEGF release through enhancing p44/p42 MAP kinase activation in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether zinc affects the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ZnSO(4) but not Na(2)SO(4). The enhancing effect of ZnSO(4) was dose-dependent between 1 and 100 muM. ZnSO(4) markedly enhanced the FGF-2-induced phosphorylation of p44/p42 MAP kinase while having little effect on the SAPK/JNK phosphorylation. PD98059 significantly reduced the amplification by ZnSO(4) of the FGF-2-stimulated VEGF release. Taken together, our findings strongly suggest that zinc enhances FGF-2-stimulated VEGF release resulting from up-regulating activation of p44/p42 MAP kinase in osteoblasts.     

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).