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The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

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The C-terminal (CT) domain of connexin43 (Cx43) is thought to be important in the control of gap junction function via: a.) CT phosphorylation-dependent control of gap junction assembly and gating, b.) interactions of CT with key regulatory binding partners. To more closely examine CT-dependent regulation, we have expressed a hemagglutinin-Cx43CT (amino acids 235-382) fusion protein in Normal Rat Kidney (NRK) cells under a tetracycline-responsive inducible promoter. Western blot analysis shows that Cx43CT expression is markedly induced by at least 48 h oftreatment with the tetracycline analogue, doxycycline. Furthermore, Cx43CT is modified within the cell, as several treatments/conditions that increase endogenous Cx43 phosphorylation induced a mobility shift in Cx43CT. Treatment with kinase activators, including epidermal growth factor (EGF) and the tumor promoting phorbol ester 12-O-tetradecanylphorbol-13-acetate (TPA), caused a shift in the mobility of the Cx43CT in a manner consistent with the mobility shift observed upon increased phosphorylation of endogenous Cx43. Similarly, Cx43CT in mitotic cells is extensively shifted, consistent with reports which show that Cx43 is phosphorylated to a unique phosphoisoform in mitotic cells. These results indicate that the Cx43CT can interact with at least some of the kinases that phosphorylate endogenous Cx43 in cells and possibly modulate the effects of kinase activation on gap junctional communication.

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Most connexins, the proteins that form gap junction channels, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Connexin phosphorylation has been investigated in a variety of ways. Some connexins show mobility shifts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis on phosphorylation. Kinase modulators can change the level of connexin phosphorylation and affect gap junctional communication levels. Metabolic labeling of cultured cells has allowed both phosphoamino acid identification and generation of phosphotryptic peptide maps. However, identification of the location of phosphorylated residues within the connexin sequence has required either targeted peptide synthesis, in vitro phosphorylation of known sites, and two-dimensional comigration studies or liquid chromatographic separation and N-terminal sequencing of peptides. In addition to these conventional methods, we discuss new applications of mass spectrometry to the identification of phosphorylated peptides and the specific residues phosphorylated within the connexin-derived peptide.

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To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

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Chondrocytes exposed to nitric oxide (NO) or antibody to Fas undergo cell death by apoptosis. This study examines structural and functional properties of chondrocyte-derived apoptotic bodies. In NO treated cartilage, the dense pericellular matrix that normally surrounds the cells is degraded and apoptotic bodies accumulate within and in the vicinity of the chondrocyte lacunae. Functional analysis shows that apoptotic bodies isolated from NO-treated chondrocytes or cartilage produce pyrophosphate. The levels of pyrophosphate produced by apoptotic bodies are increased by pretreatment of the chondrocytes with transforming growth factor beta and decreased by interleukin 1. Apoptotic bodies contain alkaline phosphatase and NTP pyrophosphohydrolase activities and can precipitate calcium. These results suggest that chondrocyte-derived apoptotic bodies express functional properties that may contribute to the pathologic cartilage calcification observed in aging and osteoarthritis.

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OBJECTIVE: To address the influence of age on inorganic pyrophosphate (PPi) accumulation in human articular chondrocytes. METHODS: Articular cartilage was obtained from men and women in 2 different age groups: ages 15-55 and 56-91. The effects of transforming growth factor beta1 (TGFbeta1) on PPi levels in the media and cell lysates of chondrocytes were investigated. In addition, the effects of TGFbeta on PPi accumulation were compared with chondrocyte proliferation. RESULTS: TGFbeta1 increased PPi levels to a greater extent in chondrocytes from subjects in the older age group compared with those obtained from younger subjects. Treatment of chondrocytes with TGFbeta1 led to a similar increase in total intracellular protein in both age groups. Although TGFbeta increased nucleoside triphosphate pyrophosphohydrolase activity and decreased alkaline phosphatase activity, these effects did not differ between the 2 age groups. Analysis of the same cell preparations showed an age-related decrease in TGFbeta-induced chondrocyte proliferation, whereas these same cells showed an increased response with respect to PPi elaboration. CONCLUSION: These results show that aging differentially affected TGFbeta-induced PPi accumulation versus proliferation in human articular chondrocytes. These differences in TGFbeta response are likely to contribute to the development of age-associated cartilage diseases such as osteoarthritis.

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The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.

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Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.

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The highly regulated enzyme HMG-CoA reductase generates mevalonate, the precursor of a complex series of isoprenoids that posttranslationally modify (isoprenylate) certain proteins (e.g., the low-molecular-weight GTP-binding proteins) or that are incorporated into cholesterol and other end products. We recently reported that isoprenoids are required for NADPH oxidase activity in granulocytes via LMW GTP-binding protein isoprenylation. In this study, we evaluated the effects of isoprenoid depletion on the expression of proinflammatory genes in human monocytic THP-1 cells. We selected conditions under which pretreatment for 24 h with isoprenoid synthesis inhibitors (HMG-CoA reductase inhibitor lovastatin or compactin at 10 microM) did not compromise cell viability but markedly suppressed H2O2 generation. Under these conditions interleukin-8 (IL-8) production was attenuated (by 50-90%) in response to lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, and phorbol myristate acetate. Coincubation of reductase inhibitor-treated cells with mevalonate prevented the attenuation of IL-8 production by reductase inhibitors. The effects of isoprenoid depletion on cytokine production were selective: IL-1 beta generation was not inhibited but the production of IL-6 and IL-8 was concomitantly suppressed. IL-8 induction was suppressed at least in part through attenuation of the increase in mRNA in stimulated cells. We conclude that isoprenoid generation through the mevalonate pathway is a requirement for IL-8 induction by activated monocytic cells in vitro. Isoprenylation inhibitors have the potential to alter monocyte proinflammatory function.

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T lymphocytes, macrophages, and oxidized low-density lipoprotein (Ox-LDL) are collocalized in early atherosclerotic lesions. Using a low-endotoxin in vitro system, we observed that Ox-LDL but not native LDL induced the production, by both freshly adherent human peripheral blood monocytes and human monocytic THP-1 cells, of the alpha chemokine interleukin (IL)-8, a potent chemoattractant for T lymphocytes. Marked IL-8 induction by Ox-LDL did not require IL-1 beta generation in THP-1 cells. Ox-LDL-induced chemokine production was selective, as Ox-LDL did not stimulate the production by THP-1 cells of the T-lymphocyte chemotactic beta chemokine macrophage inflammatory protein (MIP)-1 alpha. IL-8 induction increased in proportion to the extent of oxidation of LDL as measured by the content of lipid oxidation end products. To identify potentially active components of Ox-LDL, we tested malondialdehyde, an arachidonate-derived lipid oxidation product, and 9-hydroxyoctadecadienoic acid, an oxidation product of linoleate, the major polyunsaturated fatty acid in LDL, and observed that they induced IL-8 generation in the absence of Ox-LDL. Furthermore, when most free lipid oxidation products were removed from Ox-LDL by dialysis, some IL-8-inducing activity was released into the dialysate. However, the major IL-8-inducing activity was not dialyzable. To address the nature of the LDL particle modification required to induce IL-8, acetylated or malondialdehyde-treated native LDL particles were monitored for activity. Neither procedure rendered LDL capable of inducing IL-8. However, phospholipase A2-treated LDL induced THP-1 cell expression of IL-8.(ABSTRACT TRUNCATED AT 250 WORDS)

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