Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite.

The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

The expression of dentin sialophosphoprotein gene in bone.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are expressed as a single mRNA transcript coding for a large precursor protein termed dentin sialophosphoprotein (DSPP). DSP, DPP, and DSPP have been considered to be tooth-specific. To test for the expression of the dspp gene in bone, we performed Western immunoblots and reverse-transcription polymerase chain-reaction (RT-PCR). With Western immunoblots, we detected DSP in the Gdm/EDTA extracts of rat long bone, at a level of about 1/400 of that in dentin. Using RT-PCR, we detected DSPP mRNA in mouse calvaria. Similar to Western immunoblots, the results of RT-PCR indicated that the dspp gene is expressed at a lower level in bone than in dentin and odontoblasts. Analysis of the data shows that DSPP is not a tooth-specific protein, and that dramatically different regulatory mechanisms governing DSPP expression are involved in the bone and dentin.

Analysis of the promoter region of human cartilage oligomeric matrix protein (COMP).

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein expressed in cartilage, ligament, and tendon. The importance of COMP in the matrix of these cells is underscored by the discovery that mutations in COMP cause the skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Here, we present the first report on the analysis of the human COMP promoter region in cartilage, ligament, and tendon cells. A 1.7-kb region of the COMP promoter has been cloned and sequenced and no TATA or CAAT boxes were found. Primer extension identified multiple transcription start sites. All four transcription start sites were utilized in chondrocytes with only three of them utilized in tendon and ligament cells. Differential regulation was observed for different parts of this 1.7-kb region with the 370-bp proximal region conveying the strongest promoter activity. The highest activity was observed in tendon and ligament. Finally, we provide evidence that the DNA binding protein SP1 plays a role in the regulation of COMP expression. These results indicate that COMP expression within these cells is regulated in a unique manner that differs from the expression of other extracellular matrix genes.

Enhanced expression of osteopontin by high glucose. Involvement of osteopontin in diabetic macroangiopathy.

Atherosclerotic vascular disease is a major complication of diabetic patients. Osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin. Azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Upstream stimulatory factor 1 regulates osteopontin expression in smooth muscle cells.

Vascular smooth muscle cells (SMCs) undergo a dramatic phenotypic transition in response to injury and ex vivo culture that includes enhanced proliferation, migration, matrix deposition, and alterations in gene expression. Osteopontin is a good marker for the injury-induced SMC phenotypic state in vivo and in vitro. To identify transcription factors that might control the regulation of osteopontin expression, we investigated cultured vascular SMCs that express high and low levels of osteopontin. Using nuclear run-on assays, mRNA stability studies, and deletion analysis, we demonstrate that regulation of osteopontin steady-state mRNA levels in SMCs occurs at the transcriptional level. Transient transfection and gel-shift analyses of osteopontin promoter indicated that a region between -123 and +66 was involved in the expression of osteopontin. Supershift EMSAs identified the bHLH-leucine zipper transcription factor upstream stimulatory factor-1 (USF1) as the protein binding to this sequence. Finally, we show that USF1 protein is induced in vivo within 24 h of balloon angioplasty of rat carotids coordinately with osteopontin induction. These data suggest that USF1 governs expression of osteopontin in cultured vascular SMCs and might contribute to initial osteopontin expression observed post carotid injury and in vascular pathologies in vivo.

Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells.

Atherosclerotic vascular disease is a major complication of diabetic patients, and osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin, and azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (the key enzyme of the hexosamine pathway), profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Dual 1,25-dihydroxyvitamin D3 signal response pathways in osteoblasts: cross-talk between genomic and membrane-initiated pathways.

Osteoblasts are key regulatory cells in the control of systemic calcium ion (Ca2+) homeostasis. They, along with cells in the kidney and intestine, function as an integral part of the vitamin D endocrine system. The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), interacts with osteoblasts at several levels to modulate their phenotype and function. The interactions involve distinct receptor systems that operate on unique time scales. Rapid nongenomic actions (milliseconds to minutes), mediated through membrane receptor systems, do not require protein synthesis and include activation of voltage-sensitive Ca2+ channels, induction of phospholipid and sphingolipid turnover, elevation of intracellular Ca2+ concentrations, priming of parathyroid hormone (PTH)-sensitive ion channels, and activation of second messenger systems. In the longer term (many hours to days), interactions mediated through binding of 1,25(OH)2D3 to nuclear receptors, present in mature osteoblasts, modulate transcription of target genes. Target genes for 1,25(OH)2D3 including those encoding for the bone matrix proteins osteopontin (OPN) and osteocalcin (OCN), possess vitamin D response elements (VDRE) upstream of the transcriptional start site. In addition, it is now clear that a number of intermediate effects (1 to 3 hours) also occur. These effects require longer times than the aforementioned rapid effects, but precede the long-term consequences of activation of nuclear receptors. These include alterations in the phosphorylation state of various proteins secreted by osteoblasts, including OPN, attributable to activation/inactivation of various intracellular protein kinases and phosphatases. Intermediate effects are likely to involve both membrane-initiated rapid actions and transcriptional effects on early genes that do not require the nuclear receptor for 1,25(OH)2D3. These intermediate effects, therefore, represent ideal targets for the study of the interaction or "cross-talk" between rapid membrane-initiated, nongenomic effects and nuclear receptor-mediated, genomic effects of 1,25(OH)2D3 on osteoblasts. Intermediate effects also invite the study of rapid, non-nuclear receptor-mediated genomic effects of 1,25(OH)2D3 on osteoblasts. Complete understanding of the mechanisms by which 1,25(OH)2D3 exerts its pleiotropic effects on osteoblasts and other target cells, contributing to control of Ca2+ homeostasis, requires integration of the concepts learned from studies of both rapid and long-term pathways.

Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameloblasts, and experimentally induced odontoblast-like cells.

Dentin sialoprotein (DSP) and dentin phosphoproteins (DPP) are uniquely expressed by differentiating and fully differentiated mature odontoblasts. It is likely that DSP and DPP actively participate in the conversion of predentin to dentin. To compare the expression patterns of DPP and DSP, we constructed mouse cDNA probes. Northern analyses confirmed their specific expressions in tooth germ-derived RNA and showed that the probes reacted with similar or identical multiple transcripts. In situ hybridization indicated that DSP and DPP transcripts were uniquely detected and codistributed in developing mouse odontoblasts and preameloblasts. These data, as well as the adjacent positioning of the DSP and DPP coding sequences, suggest that common regulatory mechanisms control DSP and DPP expressions. Dental papillae cultures, in which odontoblast differentiation was experimentally induced with TGFbeta1 combined with heparin, were used to show that the two molecules are also coexpressed under in vitro conditions.

Comparison of 6-s-cis- and 6-s-trans-locked analogs of 1alpha,25-dihydroxyvitamin D3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses.

The hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] generates biological responses via both genomic and rapid, nongenomic mechanisms. The genomic responses utilize signal transduction pathways linked to a nuclear receptor (VDRnuc) for 1alpha,25(OH)2D3, while the rapid responses are believed to utilize other signal transduction pathways that may be linked to a putative membrane receptor for 1alpha,25(OH)2D3. The natural seco steroid is capable of facile rotation about its 6,7 single carbon bond, which permits generation of a continuum of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended). To identify the shape of conformer(s) that can serve as agonists for the genomic and rapid biological responses, we measured multiple known agonist activities of two families of chemically synthesized analogs that were either locked in the 6-s-cis (6C) or 6-s-trans (6T) conformation. We found that 6T locked analogs were inactive or significantly less active than 1alpha,25(OH)2D3 in both rapid responses (transcaltachia in perfused chick intestine, 45Ca2+ influx in ROS 17/2.8 cells) and genomic (osteocalcin induction in MG-63 cells, differentiation of HL-60 cells, growth arrest of MCF-7 cells, promoter transfection in COS-7 cells) assays. In genomic assays, 6C locked analogs bound poorly to the VDRnuc and were significantly less effective than 1alpha,25(OH)2D3 in the same series of assays designed to measure genomic responses. In contrast, the 6C locked analogs were potent agonists of both rapid response pathways and had activities equivalent to the conformationally flexibile 1alpha,25(OH)2D3; this represents the first demonstration that 6-s-cis locked analogs can function as agonists for vitamin D responses.

Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.

Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx.

Analogs of the seco-steroid hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)-2D3] can preferentially stimulate genomic or nongenomic signaling pathways in osteoblasts. In this study, we used 1,25-(OH)2D3 analogs and voltage-sensitive Ca2+ channel (VSCC) ligands, including dihydropyridines (Bay K 8644 and nitrendipine), in an osteosarcoma cell model to examine the relationship between 1,25-(OH)2D3-stimulated Ca2+ influx and genomic and nongenomic pathways leading to osteoblast activation. Northern blotting experiments demonstrated that an analog of 1,25-(OH)2D3, 1,24-dihydroxy-22-ene-24-cyclopropyl D3, increased messenger RNA (mRNA) levels of both osteopontin (OPN) and osteocalcin (OCN) without triggering Ca2+ influx through VSCCs. Nitrendipine (an inhibitor of L-type VSCCs) did not block the mRNA increase induced by either analog 1,24-dihydroxy-22-ene-24-cyclopropyl D3 or 1,25-(OH)2D3. 1-Deoxy analogs of 1,25-(OH)2D3, 25-hydroxy-16-ene-23-yne-D3, or 25-hydroxy-23-yne-D3, which stimulate Ca2+ influx, did not produce mRNA accumulation for OPN and OCN, consistent with their poor binding to nuclear receptors. Likewise, Bay K 8644, an agonist of VSCCs that produces Ca2+ influx, did not increase mRNA levels for OPN or OCN. Experiments using a construct derived from the sequence of the genomic OPN promoter region and a luciferase reporter confirmed the analog specificity in stimulating transcription. Together these results indicate that 1,25-(OH)2D3-mediated up-regulation of genes encoding OPN and OCN is independent of Ca2+ influx and suggest that the stimulation of Ca2+ influx by 1,25-(OH)2D3 is not required for target gene activation.

Calcitriol regulation of osteopontin expression in mouse epidermal cells.

We previously showed that calcitriol (1 alpha,25-dihydroxyvitamin D3) induces clonal mouse epidermal JB6 Cl41.5a cells to synthesize and secrete a nonphosphorylated form of osteopontin (OPN) in a dose-dependent and metabolite-specific manner. To investigate whether OPN expression is transcriptionally regulated by calcitriol in these cells, we first determined the early time course of calcitriol-induced expression of OPN protein and steady state levels of OPN messenger RNA (mRNA). Calcitriol treatment of JB6 Cl41.5a cells for 6 h caused increased secretion of [35S]methionine-labeled OPN, with maximal levels attained after 8 h of treatment. Northern analyses showed that steady state levels of OPN mRNA increase before the synthesis and secretion of OPN protein. Treatment of JB6 Cl41.5a cells with calcitriol and the transcriptional inhibitor actinomycin-D (2-250 ng/ml) indicated that calcitriol-induced accumulation of steady state OPN mRNA and secretion of OPN protein were dose dependently inhibited by actinomycin-D. These data suggest that calcitriol regulates the expression of OPN at the level of transcription. Furthermore, calcitriol increased the steady state level of OPN mRNA in a dose-dependent manner. Calcitriol-mediated increases in OPN expression were also observed in a transfection assay using a construct consisting of a portion of the promoter region of the OPN gene fused to the luciferase reporter gene. In addition, a study using 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analog that inhibits mRNA synthesis, showed that calcitriol treatment did not significantly increase the stability of OPN mRNA. These findings suggest that calcitriol increases the expression of OPN mRNA and protein by stimulating transcription.

Human submandibular gland statherin and basic histidine-rich peptide are encoded by highly abundant mRNA's derived from a common ancestral sequence.

The molecular cloning of sequences encoding human submandibular gland (SMG) statherin and a basic histidine-rich peptide is described. The corresponding mRNA's were highly abundant in the human and the Rhesus monkey (Macaca mulatta) SMG, but no homologous message was detectable in the murine SMG. Sequence analysis revealed strong homology between the statherin and basic histidine-rich mRNA's, suggesting their evolution from a common ancestral sequence.

Isolation and characterization of a mucin-glycoprotein from rat submandibular glands.

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.