Faculty Bibliography

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions

Human submandibular saliva reduces human immunodeficiency virus type 1 (HIV-1) infection in vitro. To define the mechanism of inhibition, virus was incubated with saliva or medium, velocity sucrose gradient centrifugation was performed, and fractions were analyzed for p24 and gp120. The results show that after incubation with saliva, the envelope glycoprotein was displaced from both a laboratory-adapted and a low-passage clinical HIV-1 isolate. To identify the salivary protein(s) responsible, submandibular saliva was fractionated by anion- exchange chromatography. Protein fractions containing anti-HIV activity were assayed for their ability to strip gp120 from virus. The partially purified active fractions contained two high-molecular-weight sialyated glycoproteins identified as salivary agglutinin and mucin, as well as several lower-molecular-weight proteins. It thus appears that specific salivary proteins interact with HIV-1 to strip gp120 from the virus with a resultant decrease in infectivity

The TGF-betas are multipotent in their biological activity, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription. However, TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway which involves a phosphatidylcholine-specific phospholipase and a protein kinase C. The present results, through the use of specific inhibitors of geranylgeranyl transferase I, farnesyl transferase, and acyl transferase, demonstrate that geranylgeranylated and acylated, but not farnesyslated protein(s) is required for this TGF-beta1 effect. In addition, the general tyrosine kinase inhibitor genistein completely blocked this TGF-beta1 effect. The results suggest that the TGF-beta1 signaling pathway requires not only receptor ser/thr kinase activity, but also tyrosine kinase and small GTPase activities

PURPOSE: Fibrosis of bladder tissue is characterized by an abnormal deposition of connective tissue within different layers of the bladder wall, resulting in a low volume, high pressure vesical which may ultimately contribute to renal scarring and failure. These bladders are functionally referred to as 'non-compliant' and may result from different etiologies: neurogenic, which encompasses myelodysplasia and spinal cord injury, or non-neurogenic, owing to obstruction or radiation therapy. To examine the molecular mechanisms responsible for this fibrosis, we have analyzed a well-characterized pediatric patient population for alteration(s) in collagen types I and III regulation at the protein and nucleic acid levels. MATERIALS AND METHODS: Immunohistochemical localization of collagen subtypes (I, III, and IV) was carried out using type specific monoclonal antibodies. Total collagen was determined by hydroxyproline analysis, and subtype specific expression of collagenous proteins, following cyanogen bromide extraction procedures, was quantified by competitive ELISA. Total RNA was extracted by guanidinium/phenol/chloroform, and slot blot hybridization analyses with radiolabeled human cDNA probes were quantified by densitometry of resulting autoradiograms. RESULTS: Connective tissue infiltration of detrusor smooth muscle bundles was specific for type III collagen. Protein analyses demonstrated: 1) an increase in total collagen, 2) a statistically significant increase in the type III: type I collagen ratio, and 3), an absolute increase in type III collagen protein in non-compliant bladder tissue. At the mRNA level, there was a coordinate increase in both collagen I and III steady-state mRNAs in non-neurogenic bladder tissue, whereas neurogenic bladder tissue was characterized by an increase in the type III: type I mRNA transcript ratio. CONCLUSIONS: These data suggest that regulation of collagen synthesis in bladder fibrosis is complex and is characterized by both transcriptional and post-transcriptional mechanisms, depending upon the etiology of the fibrosis

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity

Saliva is an enriched milieu containing biologically active proteins, including several different growth factors and cytokines. This study documents that vascular endothelial growth factor (VEGF), a potent, multifunctional, angiogenic cytokine, is a component of normal human saliva. VEGF was measured by ELISA in whole saliva (median concentration, 460 pg/ml) and in ductal secretions obtained from the parotid (277 pg/ml) and the submandibular-sublingual (80 pg/ml) salivary glands. VEGF seems to be synthesized endogenously by the salivary glands because both VEGF mRNA and protein (as revealed by in situ reverse transcriptase-PCR and by immunohistochemistry, respectively) colocalized to serous acinar cells and ductal epithelial cells within the parotid, submandibular, and minor salivary glands. These findings point to the existence of a 'salivary VEGF system.' It is possible that salivary VEGF plays a role in regulating physiologic and pathologic angiogenic and other vascular responses in salivary and mucosal tissues. And in particular, the presence of VEGF in saliva may contribute to the remarkable healing capacity of the oral mucosa as well as other regions of the digestive tract

The cytokine transforming growth factor-beta has multiple effects on a wide variety of cell types. These effects include modulation of growth and regulation of gene transcription. In the present work, we demonstrate that TGF-beta1 increases transcription of the latent transforming growth factor-beta binding protein-2 ( LTBP-2) gene in cultured human fetal lung fibroblasts leading to a significant increase in LTBP-2 mRNA steady state level. The stability of LTBP-2 mRNA was not appreciably altered. A corresponding increase in production of LTBP-2 protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that a member of the Ras super family and a protein kinase C, probably of the atypical (non-diacylglycerol, non-Ca++ dependent) class are likely to be components in the signaling pathway. However, phospholipases, G proteins and extracellular-signal regulated kinases do not appear to be involved. These results combined with previous findings on elastin regulation by TGF-beta1 (Kucich et al. (1997). Am. J. Respir. Cell Mol. Biol., 17: 10-16) demonstrate that TGF-beta1 can coordinately increase the steady state levels of mRNAs encoding components of the elastic fiber, but through diverse mechanisms. In contrast to LTBP-2, increased elastin expression is achieved by message stabilization. Furthermore, the TGF-beta1 signaling pathways differ and while the pathway leading to increased LTBP-2 transcription shares components with those modulating transcription of other genes, it is unlikely to be precisely congruent with any other previously described one.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization