Search

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.

Syndecan-4 (syn-4), a transmembrane heparan sulfate-containing proteoglycan, is unique among the four members of the syndecan family in its specific cellular localization to complex cytoskeletal adhesion sites, i.e., focal adhesions. During early phenotypic redifferentiation of neonatal cardiomyocytes in culture, immunolocalization reveals syn-4 to be heavily concentrated in the perinuclear endoplasmic reticulum-Golgi region, with little found at the peripheral regions. Subsequently, syn-4 becomes localized to a cytoskeletal adhesion complex unique to striated muscle, the costamere. Soon after redifferentiation of myofibrils in cultured neonatal cardiomyocytes, syn-4 is present only in costameres, not in focal adhesions. In cultured adult cardiomyocytes, it is present in both costameres and focal adhesions—the latter in two distinct regions of the spread cardiomyocytes, reflecting localization with two types of actin-containing filaments. The fact that syn-4 is observed early in the costameric regions, as opposed to later in the focal adhesions, suggests that it may play an initial role in early adhesion/signal transduction mechanisms in close proximity to the contractile apparatus, as well as in transmission of contractile force to the collagenous extracellular matrix (ECM) which surrounds the cardiac myofibers in situ. With respect to possible regulatory mechanisms of syn-4, we localized syn-4 with both the ϵ isoform of protein kinase C and the tyrosine kinase pp60csrc in costameric regions. These findings suggest that syn-4 may not only play a role in cellular adhesion and contractile force transmission, it may also, through ser, thr, and tyr phosphorylation, be part of an interactive signal transduction mechanism in myocardial functioning via these adhesive cytoskeletal complexes. Anat Rec 268:38–46, 2002. © 2002 Wiley-Liss, Inc.

Hereditary multiple exostoses (HME), a condition associated with development and growth of bony exostoses at the ends of the long bones, is caused by germline mutations in the EXT genes. EXT1 and EXT2 function as glycosyltransferases that participate in the biosynthesis of heparan sulfate (HS) to modify proteoglycans. HS proteoglycans, synthesized by chondrocytes and secreted to the extracellular matrix of the growth plate, play critical roles in growth plate signaling and remodeling. As part of studies to delineate the mechanism(s) by which an exostosis develops, we have systematically evaluated four growth plates from two HME and two solitary exostoses. Mutational events were correlated with the presence/absence and distribution of HS and the normally abundant proteoglycan, perlecan (PLN). DNA from the HME exostoses demonstrated heterozygous germline EXT1 or EXT2 mutations, and DNA from one solitary exostosis demonstrated a somatic EXT1 mutation. No loss of heterozygosity was observed in any of these samples. The chondrocyte zones of four exostosis growth plates showed absence of HS, as well as diminished and abnormal distribution of PLN. These results indicate that, although multiple mutational events do not occur in the EXT1 or EXT2 genes, a complete loss of HS was found in the exostosis growth plates. This functional knockout of the exostosis chondrocytes’ ability to synthesize HS chains further supports the observations of cytoskeletal abnormalities and chondrocyte disorganization associated with abnormal cell signaling.

Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione- S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [35S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (∼60%) and NOS2 activity (∼45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation. Cell Motil. Cytoskeleton 48:149–162, 2001. © 2001 Wiley-Liss, Inc.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of l -selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis , suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

Incubation of cultured neonatal rat cardiomyocytes in hypoxic conditions, mimicking the deprivation of O2 which occurs during in situ myocardial ischemia, leads to a progressive change in cardiomyocytes cytoskeletal components. Confocal scanning laser immunofluorescence microscopy (CSLIM) reveals that the typical striated costameric distribution of vinculin gradually disappears to be replaced by circular, vinculin-containing sarcolemmal rosettes. There is little change in distribution of vinculin in the focal adhesions or in the intercalated disks. This cytoskeletal alteration, like that observed in virally transformed fibroblasts and phorbol ester-treated skeletal myoblasts, is inhibited by genistein, a tyrosine kinase inhibitor. Increased exposure to hypoxic conditions also produces an increase in a 92-kDa collagenase which is immunolocalized only to cardiomyocytes. As with the rosette formation, genistein also inhibits the increased expression of the 92-kDa collagenase. We suggest that this cytoskeletal change with attendant release of 92 kDa collagenase may represent a defensive mechanism on the part of the cardiomyocyte to reduce damage by reducing the cellular coupling to the extracellular collagenous matrix, thereby lessening the stresses imposed by contractile forces.

Damage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4-hydroxynonenal (4-HNE), a free radical-induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4-HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin-containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4-HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4-HNE, does not involve Ca(2+)-dependent proteases. However, 4-HNE-induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes. Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.

Insulin rapidly lowers serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels in vivo. In studies reported here, HEP G2 cells were used as a model system to investigate how insulin achieves this effect. When HEP G2 cells were incubated with 100 nM insulin for 6, 14, or 24 h, IGFBP-1 protein levels in conditioned medium fell to approximately 50% of control values. This apparently was due to a fall in the rate of IGFBP-1 protein synthesis, since HEP G2 cells incorporated 46% less [35S]methionine into IGFBP-1 during a 4-h incubation with 100 nM insulin. IGFBP-1 mRNA levels were similarly affected by 100 nM insulin, falling to 45% of control values after 2 h, and to 9% of control values after 4 h of incubation with this hormone. The fall in IGFBP-1 mRNA level is consistent with data from nuclear transcription assays. HEP G2 nuclei isolated from cells that were incubated with 100 nM insulin for 2 h synthesized only approximately 1/3 the number of IGFBP-1 transcripts as did control nuclei. Further evidence that insulin decreases IGFBP-1 gene transcription comes from transient transfections using chimeric IGFBP-1 promoter-chloramphenicol acetyltransferase reporter gene constructs. IGFBP-1 promoter activity fell to approximately 50% of control values when HEP G2 cells transfected with a construct containing the first 1205 base pairs of the IGFBP-1 promoter were incubated with 100 nM insulin for 6, 14, or 24 h. Insulin lowered both IGFBP-1 protein levels and promoter activity in a dose-dependent manner. A half-maximal effect was found at approximately 1 nM insulin and a maximal effect was found at approximately 10 nM insulin in each instance. Transfections with constructs containing smaller IGFBP-1 promoter fragments showed that the region spanning from 103 to 529 base pairs 5' to the IGFBP-1 mRNA cap site was necessary to demonstrate the inhibitory effect of insulin. These studies indicate that insulin lowers IGFBP-1 protein levels, at least in part, by rapidly decreasing the rate of IGFBP-1 gene transcription, and suggest that this insulin-mediated fall in transcription is conferred through a specific region of the IGFBP-1 promoter.

Extracellular matrix components play a vital role in the determination of heart cell growth, development of spontaneous contractile activity and morphologic differentiation. In this work we studied the physical and contractile changes in neonatal rat cardiac myocytes over the first four days of growth on three different extracellular matrices. We compared commercial laminin and fibronectin, plus a fibroblast-derived extracellular matrix, which we have termed cardiogel. Myocytes cultured on cardiogel were characterized by greater cellular area and volume when compared to cells cultured on the other single-component matrices. Spontaneous contractile activity appeared first in the cells grown on cardiogel, sometimes as early as the first day post-plating, in contrast to day three in the cells cultured on laminin. Measurements of cardiac myocyte contractility i.e. percent shortening and time to peak contraction, were made on each of the first four days in each culture. Myocytes cultured on cardiogel developed maximum shortening more rapidly than the other cultures, and an earlier response to electrical pacing. Histochemical staining for myocyte mitochondrial content, revealed that the cardiogel-supported cells exhibited the earliest development of this organelle and, after four days, the greatest abundance. This reflects both a greater cell size, as well as response to increasing energy demands. Due to the increase in volume and contractile activity exhibited by the cardiogel grown myocytes, we employed calcium binding and uptake experiments to determine the comparative cellular capacities for calcium and as an indicator of sarcoplasmic reticulum development. Also whole cell phosphorylation in the presence of low detergent was assayed, to correlate calcium uptake with phosphorylation, in an attempt to examine possible increases in calcium pump number and other phosphorylatable proteins. In agreement with our physical and contractile data, we found that the cells grown on cardiogel showed a greater calcium uptake over the first four days of culture, and increased phosphorylation. However, calcium binding was not dramatically different comparing the three culture matrices. Based on our data, the fibroblast-derived cardiogel is the matrix of choice supporting earliest maturation of neonatal cardiomyocytes, in terms of spontaneous contractions, calcium handling efficiency, cell size and development of a subcellular organelle, the mitochondrion.

Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coli. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 micrograms/well compared to plasma-derived vitronectin which reached at 0.2 micrograms/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.

Tissue-cultured neonatal cardiomyocytes can be successfully maintained in culture on a variety of extracellular matrix components such as laminin, fibronectin, and interstitial collagens (Types I and III). In vivo, however, cardiomyocytes (as well as many other cells) exist in a highly complex extracellular matrix environment composed of, in addition to the above three components, other proteins, proteoglycans, and growth factors. We have developed a procedure for culturing cardiomyocytes on a naturally occurring complete extracellular matrix, Cardiogel. This substrate, synthesized by cardiac fibroblasts, contains laminin, fibronectin. Types I and III collagen, and proteoglycans. When compared to cardiomyocytes grown on laminin alone or fibronectin alone, Candiogel-supported cardiomyocytes adhere more rapidly after plating, exhibit spontaneous contractility earlier, undergo cytoskeletal and myofibrillar differentiation earlier, and grow larger than their counterparts. We suggest that their superior growth characteristics reflect the synergistic effect of numerous extracellular matrix components signals in Cardiogel transduced by the cardiomyocyte cytoskeletal elements.

The regulation of insulin-like growth factor binding protein-1 (IGFBP-1) by its ligands, IGF-I and IGF-II, was studied in continuous cultures of HepG2 human hepatoma cells. Both IGF-I and IGF-II in concentrations as low as 1–10 nmol/l caused significant suppression of IGFBP-1 protein levels. This suppression was accompanied by decreased IGFBP-1 mRNA levels occurring within 2–4 h of exposure to IGF-I or IGF-II, and by a significant decrease in IGFBP-1 promoter activity. IGF-I and IGF-II were equipotent in suppressing basal levels of IGFBP-1 protein, mRNA and promoter activity. IGF-I, IGF-II, and IGF-analogs with low IGFBP-1 affinity, (des 1–3)IGF-I and long R3IGF-I, all potently suppressed the previously characterized increase in IGFBP-1 protein levels and promoter activity induced by cAMP and theophylline. In contrast, [Leu-27]IGF-II, which interacts with the type II but not type I IGF receptor, had no effect on IGFBP-1 protein levels or promoter activity. Our data indicate that IGFBP-1 production is inhibited by its ligands, IGF-I and IGF-II, and that this effect is probably mediated at the transcriptional level. The effects of IGF-I and IGF-II apparently occur as a result of binding to the type I IGF receptor, and are similar to the previously characterized suppressive effects of insulin on IGFBP-1 transcription mediated through the insulin receptor. When considered with previous data regarding expression of IGFBP-1 and the type I IGF receptor, our results suggest that IGF regulation of IGFBP-1 may play an as yet undefined role in fetal development and postnatal hepatic regeneration.