Involvement of ErbB-2 in rheumatoid synovial cell growth.

OBJECTIVE: The synovial tissue affected by rheumatoid arthritis (RA) is characterized by hyperproliferation of synovial cells. High amounts of epidermal growth factor (EGF) in the synovial fluid of RA patients contribute to the growth of rheumatoid synovial cells. To characterize the receptor for EGF in rheumatoid synovial cells, the expression and function of ErbB family members were examined. METHODS: Synovial tissues were obtained from surgical excisions. The expression of ErbB products was examined by immunohistochemistry and immunoblotting by using specific antibodies. Primary cultures were established from the surgical materials. Cell growth was measured using MTT. The levels and phosphorylation state of the ErbB-2 protein were analyzed by immunoprecipitation and immunoblotting. RESULTS: The expression of ErbB-2, but not other ErbB-related products, was detected in synovium with RA as compared with that with osteoarthritis (OA) and ligament injury. Growth of primary synovial cells with RA was inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, a specific monoclonal antibody against ErbB-2. Herceptin showed a small effect on growth of primary synovial cells with OA. EGF stimulated the phosphorylation of ErbB-2 in primary synovial cells with RA. This EGF-stimulated phosphorylation was completely abrogated by genistein and herceptin. CONCLUSION: ErbB-2 is expressed in rheumatoid synovial cells and may function as the receptor for EGF. Our data suggest that mitotic signals from EGF family members are transduced by ErbB-2 in these cells. Inhibition of ErbB-2 may provide a new approach to the effective treatment for RA.

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation.

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal-regulated kinase was specifically activated within 1 min after UV irradiation in the range 320-360 nm. In contrast, P38 kinase was activated by 270-280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.

Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi.

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides.

It has been demonstrated that the phospholipase C-gamma (PLC-gamma) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myr-PCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The Ki values of myr-PCI(Y) and myr-PCI(F) for purified PLC-gamma1 were 3.5 and 55 microM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI(F) was much less potent than that of myr-PCI(Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.

Growth inhibition by phospholipase C inhibitor peptides of colorectal carcinoma cells derived from familial adenomatous polyposis.

We reported previously the enhanced phosphoinositide metabolism and constitutive activation of phosphoinositide-specific phospholipase C (PLC) in two colorectal carcinoma cell lines, KMS-4 and KMS-8, derived from familial adenomatous polyposis patients. To study the physiological role of enhanced PLC activity in these cells, we analyzed the effect of PLC inhibitor (PCI) peptides on their growth and cell cycle. N-Myristoylated PCI peptide, myr-PCI(Y), originally developed based on the PCI sequence of PLC-gamma 2, inhibited activity of purified PLC isoforms in vitro. When myr-PCI(Y) was added to KMS-4 and KMS-8 cultures, it suppressed the production of inositol trisphosphate, DNA synthesis, and cell growth, all of which were induced by serum in both KMS-4 and KMS-8 cells. The number of colonies grown in soft agar was also reduced significantly by treating KMS-8 cells with myr-PCI(Y) peptide. Flow cytometry analysis with propidium iodide labeling revealed marked decreases in the percentage of KMS-8 cells in S phase and increases in G0-G1 by the addition of myr-PCI(Y). On the other hand, myr-PCI(F), in which two of the tyrosine residues in myr-PCI(Y) are replaced by phenylalanine and which does not inhibit phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity in vitro, did not significantly inhibit either inositol trisphosphate production or cell growth. These results indicate that the activation of PLC is essential for growth and the transformed properties of these colorectal carcinoma cells.

Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells.

Angiotensin II (Ang II) is a potent regulator of proximal tubule functions, including transport, metabolism, and cell proliferation. The opossum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated and activated in response to various agonists. We investigated Ang II effects on serine/threonine kinase cascades in OK cells. The major findings of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it increased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(-11) M). The time course of activation was sequential; the peak stimulation was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein and herbimycin) did not inhibit AngII-induced MAPK activity. This activation of MAPK was also inhibited via AT1 receptor antagonist, Dup753 and pertussis toxin. This evidence suggests that the activation of serine/threonine cascades by Ang II is largely dependent on PMA-sensitive PKC, and is not dependent on tyrosine kinase and pertussis toxin.

AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells.

We investigated the effects of epidermal growth factor (EGF) and arginine vasopressin (AVP) on Raf-1-MAP kinase cascade, including Raf-1-kinase (Raf-1-K), MAP kinase kinase (MAPKK), MAP kinase (MAPK) and S6 kinase (S6K) in Madin-Darby canine kidney (MDCK) cells. In a dose-dependent manner (10(-10) M to 10(-6) M), EGF increased autophosphorylation of Raf-1-K and activated MAPKK, MAPK and S6K. Sequential activation of these kinases was indicated by their peak times of activation (Raf-1-K 5 min; MAPKK 10 min; MAPK 15 min; and S6K 30 min). AVP (10(-9) M to 10(-6) M) inhibited EGF-stimulated MAP kinase cascade. 8-Bromo-cyclic AMP (cAMP) could mimic the inhibitory effect of AVP on EGF-stimulated MAP kinase cascade. These results were confirmed using H-89, an inhibitor of protein kinase A (PKA) that blocked the effect of AVP on EGF-stimulated MAPK activity. We conclude that AVP inhibits EGF-stimulated Raf-1-K, MAPKK, MAPK, and S6K activity via cAMP in MDCK cells. Our results indicate that MAP kinase cascade may play an important role in integrating the effects of AVP and EGF on distal tubule function.

Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.

Enhanced phosphoinositide metabolism in colorectal carcinoma cells derived from familial adenomatous polyposis patients.

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS-4 and KMS-8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5-trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC-gamma 1 and PLC-delta 1 in KMS-4 and KMS-8 cells. Moreover, we detected the enhancement of protein-tyrosine kinase activity and tyrosine phosphorylation of PLC-gamma 1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis.

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.