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Background and Purpose: Benign and malignant fractures of the spine may have similar signal intensity characteristics on conventional MR imaging sequences. This study assesses whether in-phase/opposed-phase imaging of the spine can differentiate these 2 entities., Methods: Twenty-five consecutive patients who were evaluated for suspected malignancy (lymphoma [4 patients], breast cancer [3], multiple myeloma [2], melanoma [2], prostate [2], and renal cell carcinoma [1]) or for trauma to the thoracic or lumbar spine were entered into this study. An 18-month clinical follow-up was performed. Patients underwent standard MR imaging with an additional sagittal in-phase (repetition time [TR], 90-185; echo time [TE], 2.4 or 6.5; flip angle, 90 degrees ) and opposed-phase gradient recalled-echo sequence (TR, 90-185, TE, 4.6-4.7, flip angle, 90 degrees ). Areas that were of abnormal signal intensity on the T1 and T2 sequences were identified on the in-phase/opposed-phase sequences. An elliptical region of interest measurement of the signal intensity was made on the abnormal region on the in-phase as well as on the opposed-phase images. A computation of the signal intensity ratio (SIR) in the abnormal marrow on the opposed-phase to signal intensity measured on the in-phase images was made., Results: Twenty-one patients had 49 vertebral lesions, consisting of 20 malignant and 29 benign fractures. There was a significant difference (P < .001, Student t test) in the mean SIR for the benign lesions (mean, 0.58; SD, 0.02) compared with the malignant lesions (mean, 0.98; SD, 0.095). If a SIR of 0.80 as a cutoff is chosen, with >0.8 defined as malignant and <0.8 defined as a benign result, in-phase/opposed-phase imaging correctly identified 19 of 20 malignant lesions and 26 of 29 benign lesions (sensitivity, 0.95; specificity, 0.89)., Conclusion: There is significant difference in signal intensity between benign compression fractures and malignancy on in-phase/opposed-phase MR imaging.

Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma., (Copyright (c) 2005 Wiley-Liss, Inc.)

Cell surface adhesion receptors of the syndecan family initiate intracellular events through clustering of receptors. This crucial clustering occurs through receptor dimerization or oligomerization, which is mediated by receptor transmembrane domains. However, the exact role of the transmembrane domain during receptor activation is not fully understood. Researchers have not yet determined whether the transmembrane domain functions solely in the physical aspects of receptor clustering, or whether the domain has additional functional roles. Here we review recent advances in understanding the functionality of transmembrane domain-dependent oligomerization of syndecan cell adhesion receptor.

The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.

The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation results in reduced PKCalpha activity by inhibiting PtdIns(4,5)P2-dependent oligomerization of the syndecan-4 cytoplasmic domain. Data from NMR and gel-filtration chromatography show that the phosphorylated cytoplasmic domain (p-4L) exists as a dimer, similar to 4L, but not as higher-order oligomers. NMR analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation of the syndecan-4 cytoplasmic domain is a dramatic conformational change near the C2 region that ablates an interaction site with the PDZ domain of syntenin. Wound healing studies further suggest that syndecan-4 phosphorylation might influence cell migration behavior. We conclude that the phosphorylation (Ser183) of syndecan-4 can play a critical role as a molecular switch to regulate its functions through conformational change.

Background: Peroxisome proliferator-activated receptor (PPAR)gamma is involved mainly in adipocyte differentiation and has been suggested to play an important role in the pathogenesis of insulin resistance and atherosclerosis. We investigated the frequencies of two common polymorphisms of PPARgamma gene, exon 6 C-->T substitution and exon B Pro12Ala in healthy subjects and analyzed the correlations between the different genotypes and insulin resistance, metabolic syndrome and cardiovascular risk factors., Methods: Anthropometric measurements, fasting glucose, insulin and lipid profiles were measured in 253 Korean females. Homeostatic model assessments and quantitative insulin sensitivity check indices were calculated. Metabolic syndrome was diagnosed according to the NCEP-ATP III guidelines and the Western Pacific Region of WHO for obesity criteria for waist circumference. Polymerase chain reaction (PCR)-restriction fragment-length polymorphism and real-time PCR were performed for genotyping of the DNAs., Results: For C161T polymorphism, allele frequencies were 0.804 and 0.196 for T allele, and 0.947 for proline and 0.053 for alanine. There was no Ala12Ala homozygote in the population. No differences were seen in the mean values of age, body mass index (BMI), blood pressure, fasting blood glucose level, fasting insulin levels, HOMA and QUICKI among different genotypes when analyzed as a whole, except that subjects with Pro12Ala had significantly higher body weight than those with Pro12Pro genotype. However, mean BMI, percent body fat and weight showed significant differences between genotypes in younger age group (< or =50 years). Although overall prevalence of metabolic syndrome had no association with the genotypes, the prevalence of decreased high-density lipoprotein cholesterol component was lower in those with the T allele than in those with the CC genotype. There was no association of the genotypes with glucose tolerance status. When the subjects were divided into four groups according to the combination of the genetic alleles of the two polymorphisms, subjects having Pro12Ala and T allele, simultaneously, showed significantly higher mean weight than those without Ala allele. Pro12Ala polymorphism seems to affect body weight, similar to the previous studies, and the effect was potentiated with the presence of T allele of C161T polymorphism., Conclusions: Although either polymorphism failed to show significant association with insulin resistance, the fact that the prevalence of decreased HDL-C was lower in those with the T allele of C161T polymorphism suggests that this polymorphism might have a protective effect on atherosclerotic lipid profiles, which needs further investigation.

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Calpha activation or in syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.

Expression of syndecan-2, a transmembrane heparan sulfate proteoglycan, is crucial for the tumorigenic activity in colon carcinoma cells. However, despite the high-level expression of syndecan-2 in mesenchymal cells, few studies have addressed the function of syndecan-2 in sarcoma cells. In HT1080 fibrosarcoma cells, we found that syndecan-2 regulated migration, invasion into Matrigel, and anchorage-independent growth but not cell-extracellular matrix adhesion or proliferation, suggesting that syndecan-2 plays different functional roles in fibrosarcoma and colon carcinoma cells. Consistent with the increased cell migration/invasion of syndecan-2-overexpressing HT1080 cells, syndecan-2 overexpression increased phosphorylation and interaction of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), membrane localization of T-lymphoma invasion and metastasis gene-1 (Tiam-1), and activation of Rac. Syndecan-2-mediated cell migration/invasion of HT1080 cells was diminished when (a) cells were cotransfected with nonphosphorylatable mutant FAK Y397F or with other FAK mutants lacking PI3K interactions, (b) cells were treated with a specific PI3K inhibitor, or (c) levels of Tiam-1 were knocked down with small interfering RNAs. Furthermore, expression of several FAK mutants inhibited syndecan-2-mediated enhancement of anchorage-independent growth in HT1080 cells. Taken together, these data suggest that syndecan-2 regulates the tumorigenic activities of HT1080 fibrosarcoma cells and that FAK is a key regulator of syndecan-2-mediated tumorigenic activities.

Objective: We evaluated the association of different genotypes in C161-->T substitution in exon 6 of peroxisome proliferator-activated receptor-gamma (PPARgamma) gene with bone turnover markers, bone mineral density (BMD), and serum osteoprotegerin (OPG) in female subjects to reveal the role of PPARgamma in bone., Study Design: In 263 healthy Korean women (mean age 52 years), anthropometric measurements were taken along with measurements of lumbar spine and femoral neck BMD, bone turnover markers, such as alkaline phosphatase, serum calcium, phosphorus, 24-hour urine calcium, phosphorus excretion, and urine deoxypyridinoline. Serum follicular stimulating hormone levels were measured and serum OPG levels were measured with the enzyme-linked immunosorbent assay method. Polymerase chain reaction-restriction fragment length polymorphism was performed., Results: Allele frequencies were 0.804 for the C allele and 0.196 for the T allele. There were no differences in mean age, body mass index, BMD, and bone turnover markers among different genotypes, and the subjects with T alleles had significantly lower serum OPG levels. There were no differences in the prevalence of metabolic bone diseases according to the genotypes. When analyzed according to the menopausal status, only postmenopausal subjects with T alleles showed significantly lower serum OPG levels., Conclusion: The frequencies of C161-->T substitution in exon 6 of the PPARgamma gene in Korean females were similar to other races and women with T alleles showed significantly lower serum OPG levels and it was especially noted for postmenopausal subjects, which supports the possible concurrent association of PPARgamma and OPG with estrogen status in female subjects.

Dynamin is a large mechanochemical GTPase that has been implicated in vesicle formation in multiple cellular compartments. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. To identify potential intracellular proteins that interact with the PH domain of dynamin II, we carried out a yeast two-hybrid screen in which the PH domain of dynamin II was used as bait. The cell surface heparan sulfate proteoglycan syndecan-4 that acts in conjunction with integrins to promote the formation of actin stress fibers and focal adhesions was isolated as a binding partner for the PH domain of dynamin II. In vitro binding assays, immunoprecipitation, and confocal microscopy analysis confirmed the association of dynamin II with syndecan-4. Most dramatic finding of our study is that the cytoplasmic distribution of dynamin II and syndecan-4 changes in fibroblasts that have been stimulated to form the focal adhesions and stress fibers with LPA. In quiescent cells, dynamin II is evenly distributed in the cytoplasm and colocalizes with syndecan-4 near the nucleus. Upon treatment with LPA to induce focal adhesions and stress-fiber formation, dynamin II becomes markedly associated with syndecan-4 at focal adhesion sites. We further established the colocalization of syndecan-4 and dynamin with paxillin and actin as marker proteins for focal adhesions and stress fibers, respectively. All of these results suggest that the interaction between dynamin II and syndecan-4 is important in mediating focal adhesion and stress-fiber formation.

OPG (osteoprotegerin) is an inhibitor of osteoclastogenesis and recent work suggests it has a role in atherosclerosis. Therefore we measured serum OPG levels in patients with coronary artery disease, compared the serum OPG levels among the different groups according to the number of stenotic vessels and determined whether there was any correlation with aortic calcification, LV (left ventricular) mass index and serum CRP (C-reactive protein) levels. Subjects (n=100; mean age, 57 years) who underwent coronary angiograms were enrolled. Blood pressure, body mass index, fasting blood glucose, lipid profiles and CRP levels were measured and the LV mass indices were calculated using ECGs. Serum OPG levels were measured by ELISA. The presence of calcification in the aortic notch was checked by a chest X-ray. The subjects were divided into four groups according to the number of stenotic vessels. The mean serum OPG levels increased significantly as the number of stenotic vessels increased, and the mean serum OPG levels were higher in the group with three-vessel disease compared with the groups with no- or one-vessel disease. The mean serum CRP level was significantly higher in the group with three-vessel disease compared with the groups with no-, one- and two-vessel disease. Age and LV mass index showed significant positive correlations with serum OPG levels, although significance was lost after an adjustment for age. Serum CRP levels were positively correlated with serum OPG levels even after an adjustment for age. There were no differences in serum OPG levels according to the presence of fasting hyperglycaemia or aortic calcification. In conclusion, serum OPG level was related to the severity of stenotic coronary arteries and serum CRP levels. LV mass indices showed no significant correlation with OPG levels. The precise mechanism for the role of OPG in atherosclerosis needs to be investigated further.

Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of nuclear factor I masculine B ligand. Osteoprotegerin has been shown to be an important inhibitor of osteoclastogenesis and arterial calcification in animal models. Recently, OPG has been proposed as a link molecule between osteoporosis and arterial calcification, but the relationship between circulating OPG levels and cardiovascular disease in human populations is unclear. Thus, the aim of this study was to investigate the relationship between circulating OPG levels and cardiovascular risk factors in healthy Korean women. The subjects were 286 women aged 37 to 73 (mean +/- SD, 51.5 +/- 6.9 years). We examined blood pressure, body mass index, and waist-to-hip ratio. Serum concentrations of OPG were determined by enzyme-linked immunosorbent assay. Fasting plasma glucose levels, serum lipid profiles, insulin levels, and serum follicle-stimulating hormone (FSH) levels were determined by standard methods and homeostasis model assessment of insulin resistance was calculated. We observed a significant association between serum OPG levels and age, waist-to-hip ratio, total cholesterol, low-density lipoprotein cholesterol, and FSH levels (P < .05). Among the metabolic components, the older, obese, and hypercholsterolemic subjects had higher serum OPG levels (P < .05). However, no significant relationship was found between serum OPG levels and blood pressure and fasting plasma glucose levels. We found that mean serum OPG levels were about 11% greater in postmenopausal women (mean +/- SD, 1358.5 +/- 380.0 pg/mL) than in premenopausal women (mean +/- SD, 1228.8 +/- 407.7 pg/mL, P < .001). In multiple regression analysis with OPG as the dependent variable, serum FSH and low-density lipoprotein cholesterol levels were the significant predictor for serum OPG level (R(2) = 0.051, P < .05). In conclusion, our results show that circulating OPG levels are partly associated with cardiovascular risk factors and menopausal status in healthy Korean women. Out findings suggest that OPG may be an important paracrine factor of cardiovascular disease in the female population.

Objective: Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the RANK ligand. Moreover, OPG has been shown to be an important inhibitor of osteoclastogenesis in animal models. However, the relationship between circulating OPG levels and female bone status in human populations is unclear. In this study we undertook to investigate the relationship between circulating OPG levels and bone mineral metabolism in healthy women., Patients and Measurements: Our subjects were 287 women aged 37-73 years (mean age 51.5 years). The serum concentrations of OPG were determined by enzyme-linked immunosorbent assay (ELISA). The biochemical markers of bone turnover and FSH were measured using standard methods. Bone mineral densities at the lumbar spine and femoral neck were measured by dual-energy X-ray absorptiometry., Results: Postmenopausal women had a significantly higher mean value of serum OPG than premenopausal women (1358.5 +/- 32.5 pg/ml vs. 1228.8 +/- 33.3 pg/ml, P < 0.01). Serum OPG levels were positively correlated with age (r = 0.169, P < 0.01), as were urine deoxypyridinoline levels (r = 0.133, P < 0.05) and serum FSH levels (r = 0.187, P < 0.01) in a bivariate correlation analyses. In a multiple regression analysis, only urine calcium excretion was identified as a significant predictor for serum OPG levels., Conclusions: Circulating OPG levels were found to be associated with urine calcium excretion and menopause in healthy women. Our observations suggest that circulating OPG levels reflect an antiresorptive activity in bone, and they are related to endogenous oestrogen levels.

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).

Cytokines including IL-6 and TNF-alpha play an important role in the pathogenesis of postmenopausal osteoporosis. However, the relationship between changes in the cytokine levels and subsequent bone loss in patients undergoing a bone marrow transplantation (BMT) is unclear. A total of 46 patients undergoing an allogeneic BMT were prospectively investigated. The bone turnover markers and the serum cytokines were measured before BMT and serially after BMT. Bone mineral density (BMD) was measured before and 1 year after BMT. At 1 year after BMT, the lumbar spine BMD had decreased by 4.8%, and the total proximal femoral BMD had decreased by 12.3%. The serum IL-6 and TNF-alpha levels increased until 2 and 3 weeks after BMT, respectively. The lumbar BMD was significantly decreased as the serum IL-6 and TNF-alpha levels increased by post-BMT 3 weeks. The lumbar BMD decreased significantly as the cumulative prednisolone and cyclosporine dose increased. Patients with GVHD > or =grade II had higher lumbar bone loss than patients with GVHD

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.

The vertebrate syndecans, which make up a four-member family of small type I transmembrane heparan sulfate proteoglycans, constitute evolutionarily conserved family proteins. In particular, sequences in the transmembrane and cytoplasmic domains are a unifying feature within the family. However, the extracellular domain sequences are molecule-specific, implying that different syndecans have evolved to carry out similar, but non-identical, functions. While all four syndecans have been implicated in regulation of the cytoskeleton, their roles are clearly complex. Recent developments indicate that the closely related syndecan-2 and -4 have separable functions, though both bind a number of ligands through their heparan sulfate chains. The specification of these activities is probably core protein related, but is it due to a distinct expression pattern or molecule-specific regulatory mechanisms? Although there is not yet enough data to provide unambiguous answers, here we shall review the known functions and regulatory mechanisms of syndecan-2 and -4.

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.

During cell-matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-alpha (protein kinase C-alpha) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133-8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-alpha in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-alpha. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-alpha and enhanced the translocation of PKC-alpha into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-alpha in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-alpha immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-alpha. Taken together, these results suggest that syndecan-4 interacts with PKC-alpha in vivo and regulates its localization, activity and stability.

Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through induction of cancer-specific cellular behaviour and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Colon cancer is an excellent model system for the study of this process, since both molecular genetic and morphological changes have been well established for this disease. We recently reported increased expression of the cell surface adhesion receptor, syndecan-2, in several colon carcinoma cell lines. Indeed, increased syndecan-2 expression was necessary for tumourigenic activity, suggesting that syndecan-2 might have value as both a new diagnostic marker and a possible therapeutic target. Here, we review recent advances in understanding the role of syndecan-2 in the carcinogenesis of colon cells, and discuss a leading role for this molecule in a new era for colon cancer treatment.

Background/aims: Deoxycholic acid (DCA) has been appeared to be an endogenous colon tumor promoter. In this study, we investigated whether DCA induces nuclear factor-kappa B (NF-kappa B) activation and IL-8 expression, and tauroursodeoxycholic acid (TUDC) inhibits this signaling in HT-29 cells., Methods: After DCA treatments, time courses of NF-kappa B binding activity were determined by electrophoretic mobility shift assay (EMSA). Also, we performed Western blotting of I kappa B alpha to confirm NF-kappa B activation. Time and concentration courses of DCA-induced secretion of IL-8 were measured with ELISA in supernatants of cultured media from the cells. To evaluate the role of NF-kappa B, IL-8 levels were assessed after pretreatment with using phosphorothioate-modified anti-sense oligonucleotides (ODN). Moreover, DCA-induced secretions of IL-8 were measured after pretreatment with TUDC., Results: DCA dose-dependently induced prominent NF-kappa B binding complexes from 30 min to 8 hr and degradation of I kappa B alpha. The secretions of IL-8 were increased with DCA (50-200 micro M) treatment in a time and dose-dependent manner. Pre-incubation of the cells with TUDC (0.1-10 micro M) for 2 hours caused significant decreases in DCA induced IL-8 secretion. However, transient transfection using p50 or p65 AS-ODN showed no effect on IL-8 secretion., Conclusions: DCA may play as a colonic tumor promoter through anti-apoptotic effect of NF-kappa B activation and IL-8 expression, and DCA-induced NF-kappa B independent IL-8 expression is inhibited by TUDC.

Osteoporosis is a growing health problem in women and in men. This cross-sectional study examined the association of anthropometric, lifestyle, and hormonal factors with bone mineral density (BMD) in 152 healthy Korean middle-aged men. Smoking habits and alcohol consumption were assessed by interview. Serum testosterone and insulin-like growth factor-I (IGF-I) levels were measured by radioimmunoassay, and serum growth hormone (GH) levels were measured by immunoradiometric assay. GH stimulation tests were performed after the ingestion of 500 mg of L-dopa. BMD was measured at the lumbar spine and at the femoral neck by dual-energy X-ray absorptiometry. Of the middle-aged men, 3.9% were osteoporotic and 28.3% were osteopenic at the lumbar spine site, and 5.9% were osteoporotic and 45.4% were osteopenic at the femoral neck site. Lumbar spine BMD correlated significantly with body mass index (BMI), and femoral neck BMD correlated significantly with age, BMI, and serum IGF-I levels. The lowest quartile group for serum IGF-I levels showed the lowest femoral neck BMD. Osteoporotic men by lumbar spine BMD showed significant differences from the normal BMD group in terms of BMI and smoking habits. Also, osteoporotic men by femoral neck BMD were significantly different for mean age, BMI, and serum IGF-I levels compared with the normal BMD group. On multiple regression analysis, BMI was found to be the only independent predictor of lumbar spine BMD, whereas both BMI and serum IGF-I levels were found to be the independent predictors of femoral neck BMD. Overall, 28.3%-45.4% of middle-aged Korean men were osteopenic. We suggest that higher age, a lower BMI, current smoking history, and lower serum IGF-I levels are risk factors for lower BMD in middle-aged Korean men; however, serum testosterone levels and GH secretory capacity were not found to be correlated with BMD.

The relation between thyroid hormone changes and cytokines in bone marrow transplantation (BMT) patients has not been studied. This prospective study was designed to determine the relation between thyroid hormones and cytokine levels after BMT and their effects on the mortality. We studied 80 patients undergoing allogeneic BMT. Serum thyroid hormone parameters and cytokine levels were measured before and serially during 6 months after BMT. Serum T(3) decreased to a nadir 3 weeks post-BMT and serum T(4) was lowest at 3 months post-BMT. Serum thyroid stimulating hormone (TSH) sharply decreased to a nadir at 1 week and recovered. Serum interleukin-6 increased for 2 weeks after BMT and declined thereafter. Serum tumor necrosis factor-alpha increased for 3 weeks after BMT and declined thereafter. After 3 weeks post-BMT, both cytokine levels were negatively correlated with serum T(3) and T(4) levels. A total of 29 patients died before 1 year post-BMT and 51 patients survived longer than 1 year. Those patients who died before 1 year post-BMT had significantly lower levels of T(4) at 3 weeks, 3 and 6 months than surviving patients. In conclusion, increased levels of serum IL-6 and TNF-alpha were negatively correlated with thyroid hormone concentrations in BMT recipients suggesting the role of these cytokines in euthyroid sick syndrome.

The inhibition of histone deacetylase activity is known to induce morphological changes of transformed cells. In this study, we investigated the effect of the specific HDAC inhibitor, trichostatin A (TSA), on colon carcinoma cell lines. Treatment of human colorectal carcinoma cells, KM1214 and KM12SM, with TSA induced distinct morphological changes. Both cell lines, which normally piled up in layers without clear boundary, became more flattened, and formed monolayers with evident boundaries between cells, with concomitant increased actin filament organization. Cell-cell interaction was not affected much, based on expression level, membrane localization, and interaction of E-cadherin with beta-catenin. In contrast, syndecan-2 expression was dramatically reduced and it was correlated with the morphological changes of colon carcinoma cells. Consistently, downregulation of syndecan-2 expression by antisense cDNA clearly mimicked the morphological changes in KM12SM and reduced anchorage-independent growth of colon cancer cells. All these results indicate that reduced syndecan-2 expression correlates with TSA-induced morphological changes and reduced tumorigenic activity in colon carcinoma cells.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.

It is generally agreed that euthyroid sick syndromes (ESS) are associated with an increased production of cytokines. However, there has been scarce data on the relationship thyroid hormone changes and cytokines among the patients undergoing bone marrow transplantation (BMT). Because interleukin-8 (IL-8) has been identified as a potent proinflammatory and interleukin-10 (IL-10) as an antiinflammatory cytokine, we studied the relation between thyroid hormone parameters and these cytokines following BMT. We studied 80 patients undergoing allogeneic BMT. Serum T3 decreased to nadir at post-BMT 3 weeks. Serum T4 was the lowest at the post-BMT 3 months. Serum TSH sharply decreased to nadir at 1 week and gradually recovered. Serum free T4 significantly increased during 3 weeks and then returned to basal level. Mean levels of serum IL-8 significantly increased at 1 week after BMT. Mean levels of serum IL-10 significantly increased until 4 weeks after BMT. No significant correlation was found between serum thyroid hormone parameters and cytokines (IL-8, IL-10) after adjusting steroid doses during the entire study period. In conclusion, ESS developed frequently following allogeneic BMT and cytokine levels were increased in post-BMT patients. However, no significant correlation was found between serum thyroid hormone parameters and these cytokines.

Loss of bone mass is usually detected after bone marrow transplantation (BMT) during the early post-transplant period. However, little is known about the long-term effects of BMT on bone metabolism. We have prospectively investigated 11 patients undergoing BMT. Bone mineral density (BMD) was measured before BMT, and 1, 2, and 3 yr after BMT. Serum markers of bone turnover were serially measured before BMT and 1, 2, 3, 4, and 12 weeks, 6 months, and 1 yr after BMT. The mean change in the lumbar spine (L2-4) BMD, calculated as the percent change from the baseline to the level at 1, 2, and 3 yr was -4.7% (NS), -1.1% (NS), and +6.4% (p<0.05), respectively. The mean change in the total proximal femur BMD from the baseline to the level at 1, 2, and 3 yr was -8.5% (p<0.01), -8.7% (p<0.05) and -5.6% (p<0.05), respectively. In summary, there was little decline in lumbar BMD at 1 yr following BMT and gradual recovery until 3 yr. In contrast, femoral BMD decreased much more than the lumbar area at 1 yr and did not recover until 3 yr. The mechanism of skeletal site-selective differences in the changes of BMD needs to be elucidated.

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G(0)/G(1) cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.

To elucidate the role of focal adhesion kinase (pp125FAK) in transformation, its phosphorylation in transformed fibroblasts was compared with that of detransformed fibroblasts induced by a histone deacetylase inhibitor, trichostatin A (TSA). Inhibition of histone deacetylase activity in two different ras-transformed fibroblast lines by TSA induced a morphological change into a flattened and more spread morphology, implying detransformation. These morphological changes included increased spreading ability of transformed NIH 3T3 cells on fibronectin. Of the six tyrosine phosphorylation sites in pp125FAK, phosphorylation at position 861 (Tyr-861) was clearly decreased during detransformation by TSA. It resulted from decreased activity of Src family tyrosine kinase and/or decreased amount of Src kinase interacting with pp125FAK. Furthermore, phosphorylation of Tyr-861 was reduced substantially by the Src family kinase inhibitor, PP1, while overexpression of Src kinase increased its phosphorylation, implying that Src kinase regulates phosphorylation of pp125FAK at Tyr-861. All of these findings suggest that increased phosphorylation of pp125FAK at Tyr-861 correlates with Ras-induced transformation of fibroblasts, and TSA is able to detransform them through regulation of pp125FAK phosphorylation at Tyr-861 by an Src family kinase.

Osteoporosis is a common disease among patients undergoing transplantation and a loss of bone mass is usually detected after bone marrow transplantation (BMT), particularly during the immediate post-BMT period. Post-BMT bone loss is primarily related to gonadal dysfunction and immunosuppression. Cytokines, especially interleukin 6, play an important role in the pathogenesis of postmenopausal osteoporosis. However, the pathogenetic role of cytokines in post-BMT bone loss is unknown and data on the changes of cytokines in accordance with bone turnover markers are scarce. The aim of this study was to assess the relationship between bone turnover markers and cytokines, which are regularly sampled at peripheral blood and bone marrow before and after allogeneic BMT. This prospective study included two analyses. The first was a study of 46 BMT recipients (M/F 28/18), examining the relationship between bone turnover markers and serum cytokines that were measured before and at 1 week, 2 weeks, 3 weeks, 4 weeks and 3 months after BMT. Serum intact parathyroid hormone was measured before BMT and at 3 weeks after BMT and its relation to other cytokines and bone turnover markers was evaluated. The second analysis was a study of 14 (M/F 9/5) of 46 patients in whom bone marrow plasma cytokines [interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha)] were measured at 3 weeks after BMT. The relationship between bone marrow plasma cytokines and bone turnover markers was studied because bone marrow is the microenvironment where the real changes in bone turnover occur. Serum type I collagen carboxyterminal telopeptide (ICTP), a bone resorption marker, increased progressively until 4 weeks (peak) after BMT and then decreased thereafter. Serum osteocalcin, a bone formation marker, decreased progressively until 3 weeks after BMT and then increased thereafter. Serum IL-6 increased until 2 weeks after BMT and declined thereafter. Serum TNF-alpha increased until 3 weeks after BMT and declined thereafter. There was a significant positive correlation between serum ICTP and bone marrow IL-6 levels at 3 weeks after BMT, when a marked change in bone metabolism occurs following BMT. However, a correlation between bone turnover markers and bone marrow TNF-alpha or peripheral blood cytokines was not found. At 3 months after BMT, there was a significant negative correlation between the mean daily steroid dose and the serum osteocalcin level (r = -0.43, p < 0.05). The correlation between the Mean daily steroid dose and serum ICTP was also significant (r = 0.41, p < 0.05). Our data suggest that the progressive increase in bone resorption during the immediate post-BMT period is related to both steroid dose and the increase in bone marrow IL-6, which is a potent stimulator of bone resorption in vivo.

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3-4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30 min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase ('pp125FAK') on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.

Autoimmune diseases can be transmitted and eliminated by bone marrow transplantation (BMT). There have been several cases of autoimmune thyroid disease (AITD) occurring after BMT, but AITD remission has been rarely reported. We present four cases in which the remission or transfer of AITD occurred after an allogeneic BMT. Two patients with severe aplastic anemia (SAA) showed evidence of remission of Hashimoto's thyroiditis which they had before allogeneic BMT. One patient with SAA, which developed during treatment with propylthiouracil for Graves' disease, underwent allogeneic BMT and showed evidence of Graves' disease remission following BMT. In one patient, new AITD occurred after an allogeneic BMT from an HLA-matched sibling who already had AITD. These cases support the evidence that the immune system is newly reconstituted after BMT, and severe autoimmune disease can be an indication for BMT. To fully understand the real changes in autoimmune status after BMT, long-term prospective studies are necessary.

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20 h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8 h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

O-linked N-acetylglucosamine (O-GlcNAc) modification has been described in many proteins, including nuclear pore glycoproteins. In the present study we investigated the effect of extracellular glucose on the level of modification of nuclear pore protein p62 by O-GlcNAc. We found that exposure of cells to a high concentration of glucose caused an increased level of modification of p62 with O-GlcNAc, whereas the modification of other proteins did not change detectably. The increased O-GlcNAc modification of p62 in response to glucose required the metabolism of glucose to glucosamine. The exposure of the cells to glucosamine itself also caused increased O-GlcNAc modification, whereas mannosamine and galactosamine did not. Furthermore, changes in glucose concentrations within the physiological range induced the O-GlcNAc modification in p62 in rat aortic smooth-muscle cells, indicating that this modification of p62 might occur in an intact animal. These results imply that the ambient glucose concentration has an impact on the level of O-GlcNAc modification of proteins such as p62, and that functional changes in some of these proteins might ensue.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

It is now becoming clear that a family of transmembrane proteoglycans, the syndecans, have important roles in cell adhesion. They participate through binding of matrix ligand to their glycosaminoglycan chains, clustering, and the induction of signaling cascades to modify the internal microfilament organization. Syndecans can modulate the type of adhesive responses induced by other matrix ligand-receptor interactions, such as those involving the integrins, and so contribute to the control of cell morphology, adhesion and migration.

Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Calpha and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data from NMR and circular dichroism indicate that the cytoplasmic domain undergoes a conformational transition and forms a symmetric dimer in the presence of phospholipid activator PIP2. The solution conformations of both free and PIP2-complexed 4V have been determined by two-dimensional NMR spectroscopy and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits a twisted clamp shape having a cavity in the center of dimeric interface. In addition, it has been observed that the syndecan-4V strongly interacts not only with fatty acyl groups but also the anionic head group of PIP2. These findings reveal that PIP2 promotes oligomerization of syndecan-4 cytoplasmic domain for transmembrane signaling and cell-matrix adhesion.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKCalpha activity, and it can directly interact with the phos- phoinositide PIP2. We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKCalpha beta gamma show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCalpha beta gamma and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKCalpha beta gamma by PIP2, resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP2 and PKCalpha, localize them to forming focal adhesions, and potentiate PKCalpha activity there.

Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC with high affinity. Peptide mapping and substitution studies showed that both serines were phosphoacceptors, but each had slightly different affinity, with that of serine-197 being higher than serine-198. The efficiency of phosphorylation was concentration-dependent. At low concentrations, the cytoplasmic domain peptides were monomeric, with 2 mol/mol serine phosphorylation. At higher concentrations, however, the peptides formed dimers, with only 0.5 mol/mol phosphorylation. Concentration-dependent dimerization was not altered by phosphorylation. Phosphorylation is, therefore, dependent on the conformation of syndecan-2 cytoplasmic domain, but does not affect its oligomeric status.

The transmembrane proteoglycan syndecan-4, which is a coreceptor with integrins in cytoskeleton-matrix interactions, appears to be multimerized in vivo. Both purified and recombinant core proteins form sodium dodecyl sulfate-resistant oligomers, and we now report that a synthetic peptide corresponding to the central region of syndecan-4 cytoplasmic domain (4V) also oligomerizes. The degree of oligomerization correlates with the previously reported ability to bind protein kinase C (PKC) and regulate its activity. Only multimeric recombinant syndecan-4 core protein, but not the monomeric protein, potentiated the activity of PKCalpha, and only oligomeric syndecan-4 cytoplasmic peptides were active. Changes in peptide sequence caused parallel loss of stable oligomeric status and ability to regulate a mixture of PKCalphabetagamma activity. A synthetic peptide encompassing the whole cytoplasmic domain of syndecan-4 (4L) containing a membrane-proximal basic sequence did not form higher order oligomers and could not regulate the activity of PKCalphabetagamma unless induced to aggregate by phosphatidylinositol 4,5-bisphosphate. Oligomerization and PKC regulatory activity of the 4V peptide were both increased by addition of N-terminal cysteine and reduced by phosphorylation of the cysteine thiol group. Concentration of syndecan-4 at sites of focal adhesion formation may enhance multimerization and both localize PKC and potentiate its activity to induce stable complex formation.

During cell-matrix adhesion, both tyrosine and serine/threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface proteoglycan with heparin-binding moieties. This correlates with protein kinase C (PKC) activation, and PKCalpha can become localized to focal adhesions in normal, but not transformed, cells. PKC activation has been thought to be downstream of initial receptor-ligand interactions. We now show, however, that syndecan-4 transmembrane heparan sulfate proteoglycan and PKC co-immunoprecipitate and co-patch in vivo. The core protein of syndecan-4 can directly bind the catalytic domain of PKCalpha and potentiate its activation by phospholipid mediators. It can also directly activate PKCalpha in the absence of other mediators. This activity resides in the sequence LGKKPIYKK in the center of the short cytoplasmic domain, and other syndecans lack this sequence and PKC regulatory properties. Syndecan-4 is a focal adhesion component, and this interaction may both localize PKC and amplify its activity at sites of forming adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans.

1. To obtain further evidence for the metabolic formation of methamphetamine from famprofazone in man, concentrations of methamphetamine in plasma, as well as in urine, were measured by g.l.c. In addition, intact famprofazone and famprofazone N-oxide were analysed in the urine. 2. Methamphetamine appeared in plasma 1 h after a single 100 mg dose of the drug to two male subjects, and the concentration maintained between 24 and 44 ng/ml over 2-12 h, declining to 10 ng/ml and an undetectable level respectively after 24 h. 3. Total urinary excretion of methamphetamine over 72 h was 1.9 mg for a 25 mg dose and 2.2 mg for a 50 mg dose. After a 100 mg dose, 4.6 mg of methamphetamine was excreted over 36 h. Neither intact famprofazone nor famprofazone N-oxide were detected when the urine samples after the 100 mg dose were examined. 4. The results provide further evidence that methamphetamine is a bona fide human metabolite of famprofazone and suggest that at least 20% dose may be broken down via the pathways leading to the formation of methamphetamine. This could have significant clinical implications as the result of pharmacological activity of this metabolite.