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Biomedical subjects

S M Krane

Publications and source records attributed to S M Krane.

At least 37 records · Page 2Linked to original sources
PubMed1993

Some molecular mechanisms of glucocorticoid action.

Glucocorticoids, in excess, profoundly affect the skeleton by increasing bone resorption, decreasing bone formation and altering intestinal absorption and renal excretion of mineral ions. The mechanisms underlying these actions are complex but many involve changes in expression of genes encoding critical proteins. Interaction of the glucocorticoid with its nuclear receptor results in the induction of either positive events (transactivation) by direct interaction with cis-acting sequences, or negative (transrepression) events by repression of gene transcription and/or alteration of mRNA half-lives. An example is the inhibition by glucocorticoids of collagenase synthesis. Induction of the collagenase (procollagenase) gene by inflammatory mediators, such as interleukin-1, can be inhibited by glucocorticoid transrepression. The glucocorticoid-receptor complex binds to a protein complex AP-1; consisting of the proteins c-JUN and c-FOS) and prevents this complex from inducing activation of the procollagenase gene. These observations may be applicable to the interpretation of other glucocorticoid actions and explain some of their adverse effects on the skeleton.

Base Sequence↗
PubMed1992

1 alpha,25-(OH)2 vitamin D3 enhances expression of the genes encoding Ca(2+)-binding proteins MRP-8 and MRP-14.

Two closely related Ca(2+)-binding proteins, migration inhibitory factor-related protein (MRP)-8 and MRP-14, are synthesized under specific conditions of myeloid cell differentiation. Because 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces myeloid cell differentiation and expression of other S-100 class calcium-binding proteins, we examined the effects of 1,25-(OH)2D3 on MRP mRNA levels in human U-937 histiocytic lymphoma cells. 1,25-(OH)2D3 increased MRP-8 and MRP-14 mRNA levels in a time- and dose-dependent manner. MRP mRNA levels were maximal at 24 h and remained elevated for at least 96 h after exposure of the cells to 1,25-(OH)2D3. MRP-8 mRNA accumulation required 100- to 1,000-fold higher concentrations of 25-(OH)D3, which binds to the 1,25-(OH)2D3 intracellular receptor with 100- to 1,000-fold lower affinity. Other differentiating agents, dimethyl sulfoxide, retinoic acid, and dexamethasone, also increased levels of MRP-8 and MRP-14 mRNA. Phorbol myristate acetate enhanced MRP-14 mRNA levels to a greater extent than MRP-8 mRNA levels, suggesting differential regulation of MRP gene expression by protein kinase C. The 1,25-(OH)2D3-induced relative increase in MRP mRNA levels was not changed by a 1,000-fold reduction in extracellular [Ca2+]. Thus 1,25-(OH)2D3 is potentially a physiological modulator of MRP gene expression. Expression of the MRP-8 and MRP-14 genes may be important for differentiation of myeloid cells.

Calcitriol↗
PubMed1992

Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line.

A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of type I collagen: effect on susceptibility to collagenase.

A genetic approach to define the role of collagenase in physiological and pathological bone remodeling is to identify spontaneous mutations in the collagenase gene which alter enzymatic activity. Alternatively it is possible, though site-directed mutagenesis, to alter genes encoding critical amino acid sequences in the collagen substrate, in a manner analogous to the successful development of animal models for osteogenesis imperfecta. We have thus utilized this approach to alter the Col1a1 gene to encode amino acid substitutions in sequences around the known collagenase cleavage site (glycine-isoleucine at positions 775-776) in type I collagen, and transfect these genes into homozygous Mov-13 fibroblasts, in which the endogenous Col1a1 gene is inactive. Nonconservative substitutions of proline for isoleucine at the P1' site and double substitutions of proline for glutamine (P2) and alanine (P2') resulted in type I collagen resistant to hydrolysis by collagenase. Furthermore, in normal fibroblasts transfected with a mutant Col1a1 gene encoding collagenase resistance in which an additional methionine substitution at position 776 provided a marker for the mutant protein, mutant and wild type triple helical molecules were synthesized and secreted as heterotrimers. A single mutant alpha 1(I) chain did not prevent cleavage of the wild type alpha 1(I) chain but it is likely that the uncleaved alpha 1(I) chain would prevent dissociation of the triple helical fragments containing the other cleaved chains. Introduction of these genes into transgenic mice should result in abnormal phenotypes characterized by altered connective tissue remodeling.

Amino Acid Sequence↗
PubMed1991

An interleukin 1 beta point mutant demonstrates that jun/fos expression is not sufficient for fibroblast metalloproteinase expression.

Substitution of glycine for arginine at position 127 of the mature human interleukin 1 beta protein generates a mutant IL-1 beta protein (IL-1 beta R----G) which binds cellular IL-1 receptors with high affinity but fails to elicit significant proliferation of T-helper cells (Gehrke, L., Jobling, S. A., Paik, L. S. K., McDonald, B., Rosenwasser, L. J., and Auron, P. E. (1990) J. Biol. Chem. 265, 5922-5925). Although both IL-1 beta and the IL-1 beta R----G mutein stimulate transcription of fibroblast immediate early (fos and jun) and early (IL-1 beta and IL-6) genes, the IL-1 beta R----G mutein, in contrast to the wild-type IL-1 beta protein, induces minimal or no transcription of late genes such as procollagenase and prostromelysin. The effect of the naturally occurring IL-1 receptor antagonist protein (IL-1ra) on fibroblast transcription is distinct from that of the IL-1 beta R----G mutein, for the IL-1ra fails to stimulate not only late (procollagenase and prostromelysin) but also immediate early (fos and jun) gene expression. These data suggest that the IL-I beta R----G mutein triggers an incomplete or defective signal transduction cascade and demonstrate that fibroblast fos and jun expression is not necessarily accompanied by increased transcription of genes containing the AP-1 binding site. These data also suggest that at least two events are required for IL-1-mediated late gene induction in fibroblasts.

Blotting, Northern↗
PubMed1991

Enhancement of human immunodeficiency virus 1 replication in monocytes by 1,25-dihydroxycholecalciferol.

Human immunodeficiency virus (HIV) expression and replication are under tight regulatory control. We demonstrate that 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances the replication of monocyte- and lymphocyte-tropic strains of HIV-1 up to 10,000-fold in monocyte cell lines, peripheral blood monocytes, and unfractionated peripheral blood mononuclear cells. 1,25(OH)2D3 is therefore one of the most potent regulators of HIV-1 replication described to date. Precursors of 1,25(OH)2D3 enhance HIV-1 replication in proportion to their affinity for the 1,25(OH)2D3 intracellular receptor, suggesting that 1,25(OH)2D3 influences HIV-1 replication by mechanisms involving this receptor. These studies may have important implications for the design of effective therapy of HIV-1 infection.

Antigens, CD↗
PubMed1990

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide: the role of calcium and heat shock.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We therefore examined whether 1,25-(OH)2D3 would also modulate the susceptibility to H2O2-induced oxidative stress. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) produced unexpected increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury, we investigated the effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity, as well as the effects of 1,25-(OH)2D3 and H2O2 on cytosolic-free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca2+ as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, p less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25-(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2(+)-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus, 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The protective effects of heat shock are probably exerted at a site distal to the toxic effects of Ca2+. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2(+)-associated enhanced susceptibility of oxidative injury due to 1,25-(OH)2D3.

Calcitriol↗
PubMed1990

Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene.

Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.

Amino Acid Sequence↗
PubMed1990

Mechanisms of matrix degradation in rheumatoid arthritis.

In the inflammatory synovium production of collagenase is probably responsible for the degradation of collagen in the extracellular matrix and distortion of the architecture and function of the joints. Major collagenase-producing cells are mesenchymal cells such as fibroblasts and chondrocytes, which synthesize and secrete the enzyme influenced by the action of cytokines produced by adjacent mononuclear cells. The cytokines act primarily through cell-surface receptors, whose signal is probably then mediated by complexes of nuclear oncoproteins, to activate transcription of the procollagenase gene. The increased production of collagenase ultimately is the result of a cascade of cellular effects involving complex interactions of different ligands in a system characterized by amplification and feedback loops.

Arthritis, Rheumatoid↗
PubMed1990

Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts.

Fibroblasts cultured from normal human dermis are heterogeneous with respect to growth kinetics, synthetic function, and morphologic features. There are many examples of clonal heterogeneity in apparently homogeneous connective tissue cell populations, and it has been suggested that selection of cell populations with particular phenotypic features is the basis for the development of pathologic connective tissue changes in inflammatory disorders. In these studies we report characterization of the pattern of matrix biosynthesis and responses to hormones in cells cloned from normal human dermis. The results indicate that cloned dermal fibroblasts are heterogeneous with respect to synthesis of collagens as well as their responses to prostaglandin E2 and parathyroid hormone. Selective expansion of clonal populations with unique patterns of matrix synthesis and cell surface receptors could provide the basis for abnormal connective tissue remodeling in certain pathologic states.

Clone Cells↗
PubMed1990

Clinical implications of cartilage metabolism in arthritis.

The ability of articular cartilage to withstand repeated mechanical loading with relatively little wear over a lifetime results from the properties of the extracellular matrix (ECM) and the optimal function of the chondrocytes which are responsible for the synthesis and presumably maintenance of this ECM. The properties of the ECM are accounted for by the relationship of the major aggregating, polyanionic, negatively charged proteoglycans with their potent viscoelastic properties to the network of collagens and several noncollagenous proteins. The major collagen (type II) interacts with type IX collagen in a highly specific manner. Type IX collagen has a chondroitin sulfate side chain and can also bind to the aggregating proteoglycans through a basic amino terminal domain. In inflammation, injury and probably repeated wear, function of the chondrocytes is disturbed, mediated by the action of potent cytokines, which results in release of degradative enzymes and alterations in the pattern of synthesis of the ECM. Identification of the critical cytokines and the sequence of events that result from their action should provide the basis for rational prophylaxis and therapy of disorders such as osteoarthritis and rheumatoid arthritis. Articular cartilage has unique mechanical properties which permit repeated mechanical loading with relatively little wear over a lifetime. These properties result from the special character of the extracellular matrix (ECM) and optimal functioning of the component cells (chondrocytes) which are responsible for the synthesis and presumably, maintenance of this matrix. Articular chondrocytes survive and perform these critical functions in an anaerobic environment remote from the vasculature and must derive their nutrition from the synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Increases in levels of procollagenase messenger RNA in cultured fibroblasts induced by human recombinant interleukin 1 beta or serum follow c-jun expression and are dependent on new protein synthesis.

The protein encoded by the protooncogene c-jun, included in the activator protein-1 (AP-1) complex, is probably the critical trans-acting factor controlling transcription of the procollagenase gene which is rate limiting for subsequent synthesis of procollagenase. Therefore, to elucidate possible mechanisms whereby IL-1 stimulates procollagenase synthesis, we measured levels of c-jun and procollagenase mRNA in human serum-starved dermal fibroblasts in response to human recombinant IL-1 beta (hrIL-1 beta). hrIL-1 beta or serum induced rapid increases in c-jun mRNA levels; mRNA levels declined rapidly after hrIL-1 beta and more slowly after exposure to serum. The increases in levels of c-jun mRNA preceded the increases in procollagenase mRNA. Whereas the increases in levels of procollagenase mRNA were blunted by cycloheximide, those of c-jun mRNA were enhanced. We interpret these results as follows: IL-1 or serum induce transcription of c-jun by mechanisms independent of new protein synthesis; c-JUN, the protein product of c-jun in the AP-1 complex, is an essential mediator of the effects of IL-1 or serum in the subsequent induction of expression of the procollagenase gene.

Blood Physiological Phenomena↗
PubMed1988

Cytokines.

During physiological and pathological skeletal remodelling, immune cells and stromal fibroblasts near active bone-forming and bone-resorbing surfaces might modulate the functions of skeletal tissue cells. Osteoblasts, osteoclasts and their progenitor cells are the probable direct targets of these effector cells (e.g. lymphocytes and monocytes) which act through direct contact or the release of soluble ligands (e.g. interleukin 1 or tumour necrosis factor, lymphotoxins, transforming growth factors). These cytokines bind to specific cellular receptors, resulting in changes in the form and function of the target bone cells and variable activation of genes coding for extracellular matrix proteins and proteinases which are responsible for remodelling the matrix. The synthesis and release of eicosanoids such as prostaglandins (e.g. PGE2) are frequent associated events. PGE2, in turn, affects several functions of the skeletal tissue cells as well as the lymphocytes and monocytes in their environment. The mesenchymal cells may also be induced to release ligands such as colony-stimulating factors, other cellular products or hormones resulting in a system of feedback and amplification loops. The cellular responses are thus subject to multiple controls not only determined by these ligands acting on their respective receptors but also by the pathways of signal transduction and how they, in turn, are influenced by interactions with molecules within the cells.

Animals↗
PubMed1988

Changes in serum levels of type I and III procollagen extension peptides during infusion of human parathyroid hormone fragment (1-34).

Parathyroid hormone (PTH) inhibits collagen synthesis in vitro, in organ or cell culture and cell-free translation systems. We have designed studies to measure the effects of PTH on collagen synthesis in vivo in humans, utilizing measurements of the serum levels of procollagen extension peptides during and after infusion of synthetic human PTH (hPTH) fragment (1-34). Radioimmunoassays for the carboxy-terminal peptide of type I procollagen (pColl-C) and the amino-terminal peptide of type III procollagen (pColl-III-N) were used to measure acute changes in serum during and after hPTH(1-34). In all six osteoporotic subjects and two normal individuals, serum levels of pColl-I-C were decreased by 16 hr of infusion and returned towards normal 14 hr after the infusion was discontinued; serum levels of pColl-III-N did not change significantly during the infusion, but were increased at 14 hr after the infusion was discontinued. The PTH-induced decrease in levels of pColl-I-C correlated with an increase in blood levels of ionized calcium. In all but two subjects the serum levels of 1,25-dihydroxy vitamin D [1,25(OH)2D] were also increased during the period when serum levels of pColl-I-C were decreased. These results are compatible with the conclusion that infusion of PTH acutely inhibits type I (bone) collagen synthesis, but not type III collagen synthesis. These effects could be direct or indirect, related in part to PTH-induced increased 1-alpha-hydroxylation of 25-(OH) vitamin D and the resultant increased serum levels of 1,25(OH)2D.

Adult↗