A drop in the circulating concentrations of soluble receptor for advanced glycation end products is associated with seroconversion to autoantibody positivity but not with subsequent progression to clinical disease in children en route to type 1 diabetes.

BACKGROUND: Advanced glycation end products (AGEs) and their interaction with the receptor for AGEs (RAGE) have been studied for their role in the pathogenesis and complications of type 1 diabetes. Decreased concentrations of soluble RAGE (sRAGE) have been reported in acute autoimmune inflammation. We set out to analyze the changes in sRAGE concentration during preclinical diabetes in children seroconverting to islet autoantibody positivity. METHODS: We measured serum concentrations of sRAGE in 168 children who progressed to clinical disease and 43 children who turned positive for at least 2 diabetes-associated autoantibodies but remained nondiabetic. We analyzed the sRAGE before seroconversion in the first autoantibody-positive sample and annually thereafter until the diagnosis of type 1 diabetes or end of follow-up. RESULTS: Both groups had similar sRAGE before seroconversion, but subsequently, sRAGE concentrations were lower (P < .001) in the progressors. The progressors had significantly higher sRAGE concentrations before than after seroconversion (P < .001). The nonprogressors did not experience a similar decrease. The sRAGE concentrations remained stable after seroconversion in both groups. CONCLUSIONS: These data indicate that sRAGE may be involved in the initiation of beta-cell autoimmunity but not in the progression from beta-cell autoimmunity to clinical disease.

R705H mutation of MYH9 is associated with MYH9-related disease and not only with non-syndromic deafness DFNA17.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disease caused by mutation of MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (NMMHC-IIA). MYH9-RD patients have macrothrombocytopenia and granulocyte inclusions (pathognomonic sign of the disease) containing wild-type and mutant NMMHC-IIA. During life they might develop sensorineural hearing loss, cataract, glomerulonephritis, and elevation of liver enzymes. One of the MYH9 mutations, p.R705H, was previously reported to be associated with DFNA17, an autosomal dominant non-syndromic sensorineural hearing loss without any other features associated. We identified the same mutation in two unrelated families, whose four affected individuals had not only hearing impairment but also thrombocytopenia, giant platelets, leukocyte inclusions, as well as mild to moderate elevation of some liver enzymes. Our data suggest that DFNA17 should not be a separate genetic entity but part of the wide phenotypic spectrum of MYH9-RD characterized by congenital hematological manifestations and variable penetrance and expressivity of the extra-hematological features.

Autoantibodies against zinc transporter 8 are related to age, metabolic state and HLA DR genotype in children with newly diagnosed type 1 diabetes.

BACKGROUND: We set out to define the characteristics of humoral autoimmunity against ZnT8 in children and adolescents with newly diagnosed T1D in relation to age and metabolic status at diagnosis, human leucocyte antigen (HLA) genotype and family history of T1D. METHODS: A total of 2115 subjects <15 years of age were analysed for antibodies against zinc transporter 8, ICA, GADA, IAA, IA-2A, HLA DR-DQ genotype, blood pH, plasma glucose and ß-hydroxybutyrate concentrations. Their family history of T1D was also recorded. RESULTS: Zinc transporter 8 antibodies (ZnT8A) were detected in 63% of the cases. ZnT8A positivity was associated with older age at diagnosis (mean 8.2 years versus 7.5 years, p < 0.001). Seven subjects (0.3%) had ZnT8A as their single autoantibody. Diabetic ketoacidosis at diagnosis was less common among subjects with ZnT8A than among those without (16% versus 20%, p = 0.012). The prevalence of ZnT8A was decreased in DR3/DR4 heterozygotes when compared with those with other DR combinations (p < 0.001). Subjects with the neutral DR13-DQB1*0604 haplotype tested more frequently positive for ZnT8A than the rest of the population (p < 0.001). A positive family history of T1D showed no association with ZnT8A prevalence or levels. CONCLUSIONS: Antibodies for ZnT8 is related to age and metabolic status at diagnosis as well as HLA genotype but does not significantly improve the detection rate of ß-cell autoimmunity in Finnish children and adolescents affected by T1D.

Vitamin D(3) analogs (MC 1288, KH 1060, EB 1089, GS 1558, and CB 1093): studies on their mechanism of action.

Selected 20-epi and 20-normal vitamin D(3) analogs were studied. First, point mutations were introduced into human vitamin D receptor (VDR) to identify residues important for ligand binding. In helices three, four and five, His229, Asp232, Ser237 and Arg274 seem to have an important role in the binding of calcitriol. Surprisingly, the 20-epi analog MC 1288 did not bind to Ser237. Second, the effects of analogs on VDR degradation were studied. The transcriptionally active 20-epi analogs protected VDR against degradation more efficiently than the 20-normal analogs and calcitriol. With proteasome inhibitor MG-132 formation of Sug-1-RXRbeta-VDR-VDRE complex was detected. The 20-epi analogs effectively prevented its formation. Thus, the 20-epi analogs induce a VDR conformation, which prevents binding of factors mediating VDR degradation. Third, the analogs were found to be powerful regulators of cell cycle progression in MG-63 cells. They arrested cell cycle in the G0/G1 phase at lower concentrations and earlier time points than calcitriol. This was accompanied by hypophosphorylation of Rb followed by strong inhibition of Cdk2 activity. This correlated with increased levels of p27. Cdk2 and cyclin E levels were downregulated but those of p21 and cyclin D1 were not affected. Thus, a similar sequence of events with calcitriol and the analogs in inhibiting MG-63 cell growth was detected but the analogs had much longer lasting and stronger effects than calcitriol. A unifying scheme for the varying effects of vitamin D(3) analogs is presented.

State of methylation of the human osteocalcin gene in bone-derived and other types of cells.

DNA methylation is a general mechanism of controlling tissue-specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone-derived (MG-63, U2-Os, SaOs-2) and other types (normal lymphocytes, A-498, Hep G2) of cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG-63 and induced in SaOs-2 but not in U2-Os osteoblast-like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single-copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A-498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5-azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG-63 cells. In gel mobility shift assays, human vitamin D receptor and the AP-1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression.

Synthetic 20-epi analogs of calcitriol are potent inducers of target-gene activation in osteoblastic cells.

We have compared the actions of calcitriol and its three synthetic analogs, 20-epi-22-oxa-24a,26a,27a-trihomo-1 alpha,25-dihydroxyvitamin D3 (KH 1060), 1 alpha,24S-(OH)2-22-ene-26,27-cyclopropyl vitamin D3 (MC 903) and 20-epi-1 alpha,25-dihydroxyvitamin D3 (MC 1288), on the expression of two marker genes of differentiated osteoblasts, namely alkaline phosphatase and osteocalcin, using human MG-63 osteosarcoma cells. Calcitriol and the analogs had qualitatively similar stimulatory effects on target-gene activation. Quantitatively, MC 903 behaved in most experiments essentially as the parent compound calcitriol. In vitamin D receptor/DNA complex formation MC 903, however, was more potent than calcitriol. In contrast, the 20-epi analogs, KH 1060 and MC 1288, were much more potent even at lower concentrations, than calcitriol and MC 903 in stimulating alkaline phosphatase activity, osteocalcin mRNA synthesis and osteocalcin secretion. The stimulation occurred to a greater degree and for a longer period than with calcitriol. This effect was apparently mediated by stronger and longer lasting binding of the vitamin D receptor to the osteocalcin vitamin D responsive element by the 20-epi analogs. After a 6-h treatment and during subsequent culture without hormone, the effects of the 20-epi analogs were also stronger and lasted longer than those with calcitriol or MC 903. Collectively, at comparable and lower concentrations, the 20-epi analogs, KH 1060 and MC 1288, mediate much stronger and longer lasting stimulatory effects than calcitriol or its analog MC 903 on target-gene expression associated with the differentiated phenotype of the MG-63 human osteosarcoma cells.

Mechanism of action of superactive vitamin D analogs through regulated receptor degradation.

We and others have previously shown that selected vitamin D analogs potentiate the vitamin D receptor (VDR) mediated transcription much more efficiently than the natural hormone itself. Here we show that the transcriptionally active 20-epi analogs, namely KH 1060 and MC 1288, protect VDR against degradation more efficiently than calcitriol at 10(-10) M concentration (VDR t(1/2) > 48 h, 17 h, and 10 h, respectively). The conformationally epi-like analog EB 1089 did not significantly alter the half-life of VDR (10.3 h), but retained the VDR levels over longer periods of time than calcitriol. The transcriptionally weak analog GS 1558, on the other hand, enhanced VDR degradation even more than what was observed with the unliganded receptor (t(1/2) 4.5 h and 5 h, respectively). Inhibition of proteasome activity by the inhibitor MG-132 resulted in a marked increase in the VDR levels in cells treated with the vehicle or GS 1558 (2.5-fold and 2.7-fold, respectively), more than twice the levels observed in the presence of calcitriol or EB 1089 (1.2-fold and 1.1-fold, respectively). MG-132 treatment did not increase the VDR levels in cells treated with KH 1060 or MC 1288. The electrophoretic mobility shift assay (EMSA) with nuclear extracts from MG-132-treated cells revealed formation of a high-molecular-weight RXRbeta-VDR-VDRE complex, which also contained Sug1. In the presence of calcitriol, 34% of total VDR in its DNA binding state was present in this complex. The 20-epi analogs effectively prevented the formation of this complex, since, in this case, only 16% of total VDR was found in this complex. These results suggest that KH 1060 and MC 1288 induce a VDR conformation, which prevents binding of proteins mediating receptor degradation. As a result, the regulation of VDR degradation differs from that found with the calcitriol-VDR complex resulting in superactive transcriptional action of the analogs.

Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093).

1,25-Dihydroxyvitamin D3 (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D3 analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo+ ++-1alpha,25(OH)2D3), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 microM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRbeta proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRbeta-complex with additional nuclear proteins.

Structure-function studies of new C-20 epimer pairs of vitamin D3 analogs.

A growing number of calcitriol (1alpha,25-dihydroxyvitamin D3) analogs have become available in recent years. Many of these analogs exhibit lower calcemic effects than calcitriol and inhibit cell proliferation and enhance cell differentation more efficiently than calcitriol. We have compared structure-function relationships of a series of new C-20 epimer (20-epi) vitamin D3 analogs with their natural C-20 counterparts. In human MG-63 osteosarcoma cells, quantification of cellular osteocalcin mRNA levels by Northern blot analysis and osteocalcin biosynthesis by radioimmunoassay indicated that most studied analogs at a concentration of 10 nm induced osteocalcin gene expression more efficiently than the parent compound, calcitriol. Interestingly, when the biological responses were compared with the binding affinities of the analogs to in-vitro translated human vitamin D receptor and with their ability to protect the receptor against partial proteolytic digestion, significant correlations were not observed. Further, molecular modelling of the compounds by energy minimization did not reveal marked differences in the three-dimensional structures of the analogs. These results suggest that higher than normal ligand binding affinity or 'natural' conformation of the ligand-receptor complex are not necessarily required for the 'superagonist' transactivation activity. The mechanism of action of the efficient analogs may involve stabilization and/or differential binding of transcriptional coactivators or transcription intermediary factors to the hVDR during transactivation.

Characterization of mixed monolayers of phosphatidylcholine and a dicationic gemini surfactant SS-1 with a langmuir balance: effects of DNA.

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N;N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (pi-A) curves and determined the elastic area compressibility modulus (C) as a function of lateral packing pressure and the mole fraction of the cationic lipid (X(SS-1)), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (X(SS-1) < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P(-)-N(+) dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X(SS-1) and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P(-)-N(+) dipole is further supported by the pronounced increase in monolayer dipole potential psi due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 microM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X(SS-1) < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in DeltaA/molecule, was observed at X(SS-1) > 0.5. At a higher concentration of DNA (1.88 microM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X(SS-1) = 0.5, condensation was observed at all values of X(SS-1).

Functional interference between AP-1 and the vitamin D receptor on osteocalcin gene expression in human osteosarcoma cells.

The binding of transcription factor AP-1 and vitamin D receptor (VDR) to the composite AP-1 plus vitamin-D-responsive promoter region (AP-1 + VDRE) of the human osteocalcin gene was characterized in osteocalcin-producing (MG-63) and non-producing (U2-Os, SaOs-2) human osteosarcoma cell lines. In mobility-shift assays with AP-1 + VDRE, AP-1, and VDRE probes and nuclear extracts from these cells, one AP-1-specific and two VDR-specific (fast and slow mobility) interactions were observed. Characterization of the complexes indicated that AP-1 and VDR do not bind simultaneously to the AP-1 + VDRE oligonucleotide. Intensity of the complexes was greatly influenced by cell density: in MG-63 and SaOs-2 cells, AP-1 binding was strong during the proliferative period disappearing at confluency whereas, in U2-Os cells, AP-1 binding was prominent also at the confluent stage. Furthermore, MG-63 cells possessed the faster migrating VDR complex at all stages of confluency whereas, in U2-Os and SaOs-2 cells, it was very weak or absent. There were no detectable differences in the levels of VDR protein between these cell lines. In U2-Os cells, the level of c-jun mRNA was higher than in the other two cell lines, whereas none of these cell lines exhibited detectable levels of c-fos mRNA at the confluent stage. Exogenous c-Jun protein effectively blocked the VDR-DNA interaction. Further, all these cell lines expressed mRNA for retinoid X receptor alpha (RXR alpha), the factor suggested to be required for the VDR-DNA interaction. The presence of an accessory factor in the VDR-DNA complexes was indirectly shown by treatment of the cells with 9-cis retinoic acid and by cycloheximide. Both treatments reduced VDR binding without affecting the VDR protein level. These results suggest that AP-1 interferes with VDR binding to the AP-1 + VDRE element and that the vitamin D responsiveness of the osteocalcin gene correlates with weak AP-1 binding and strong binding of the faster migrating VDR complex.

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence.

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.

Assay of collagenase activity by a rapid, sensitive, and specific method.

A new method for the assay of collagenase activity has been developed, whereby the collagen cleavage products, after initial collagenase digestion, are degraded further by a mixture of trypsin and alpha-chymotrypsin. The degradation products are soluble in TCA and can be conveniently separated from the remaining uncleaved collagen substrate by rapid filtration. The enzyme assay is shown to be reproducible and sensitive, and it lends itself to a convenient and rapid determination of collagenase activity in relatively large numbers of samples. The applicability of this method is demonstrated by the detection of increased collagenase activity in skin fibroblast cultures derived from a patient with recessive dystrophic epidermolysis bullosa.

The production of collagenase by adherent mononuclear cells cultured from human peripheral blood.

Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)