Faculty Bibliography

Transforming growth factors (TGFs)-beta are multipotent in their biologic activity, regulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription, but TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway that likely involves a phosphatidylcholine-specific phospholipase C, a protein kinase C, prenylated and acylated protein(s), and one or more tyrosine kinases. However, there is a 4- to 6-h lag period after the addition of TGF-beta1 before significant stimulation of elastin expression is observed and the question of whether the Smads are involved has not been addressed. In the present work, using cultured human fetal lung fibroblasts, we show through the use of specific inhibitors and transfection of a Smad 7 construct that in addition to de novo protein synthesis and active Smads, the extended activity of protein kinase C (PKC)-delta and the stress-activated protein kinase, p38, is required for TGF-beta1 to achieve elastin mRNA stabilization

It is possible that many of the fibrogenic effects of transforming growth factor-beta (TGF-beta) are mediated by connective tissue growth factor (CTGF). In the present work, we show that TGF-beta1 produces a 5- to 6-fold increase in CTGF expression by cultured human lung fibroblasts that is due mainly to increased transcription. The half-life of CTGF mRNA is 1.96 h, consistent with its role as a cytokine. In addition to requiring Smad activity, based upon the effects of specific inhibitors, the TGF-beta intracellular signaling pathway requires the activity of a phosphatidylcholine-specific phospholipase C, a protein kinase C, and one or more tyrosine kinases. It is also likely that the pathway requires a member of the Ras superfamily of small GTPases, but not trimeric G proteins. Pharmacologic inhibition of TGF-beta stimulation of CTGF expression may be an effective therapeutic approach to a variety of undesirable fibrotic reactions

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes

OBJECTIVE: To examine the effects of specific inhibition of geranylgeranyl transferase I on the expression of types I and III collagen genes in normal and systemic sclerosis (SSc) dermal fibroblasts in vitro. METHODS: Fibroblasts from 2 normal subjects and 4 SSc patients were incubated with 2-10 microM of GGTI-298, a specific geranylgeranyl transferase inhibitor. Type I collagen and fibronectin production were determined by enzyme-linked immunosorbent assay. Steady-state messenger RNA (mRNA) levels for alpha1(I), alpha2(I), and alpha1(III) collagens and fibronectin were assessed by Northern hybridization, and the transcription of the alpha1(I) collagen gene was examined by transient transfections with a reporter construct containing -5.3 kb of the gene. RESULTS: GGTI-298 caused a dose-dependent inhibition of type I collagen production and a reduction in the steady-state levels of alpha1(I), alpha2(I), and alpha1(III) mRNA in normal and SSc cells. A 60-70% inhibition of type I collagen production and a 70-80% reduction in the mRNA levels for alpha1(I), alpha2(I), and alpha1(III) were observed at 10 microM GGTI-298. In contrast, the expression of fibronectin, cyclooxygenase 1, and GAPDH was not affected. The effects on alpha1(I) collagen mRNA resulted from a profound reduction in transcription of the alpha1(I) collagen gene promoter. GGTI-298 did not affect cellular viability or morphology. CONCLUSION: These results demonstrate that specific inhibition of geranylgeranyl prenylation causes a potent and selective inhibition of expression of the genes encoding types I and III collagens, without affecting cellular viability. The findings indicate that inhibition of geranylgeranyl prenylation should be further studied as a potential therapeutic approach for SSc and other fibrosing diseases

The cytokine transforming growth factor-beta (TGF-beta) has multiple effects on a variety of cell types, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. In the present work, we demonstrate that TGF-beta1 produces a fourfold increase in transcription of the fibronectin gene in cultured human fetal lung fibroblasts with only a small increase in mRNA stability resulting in a significant increase in fibronectin mRNA steady state level. A corresponding increase in production of fibronectin protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that geranylgeranylated, but not farnesylated or acylated protein(s), protein kinase C-delta, phosphatidylcholine-specific phospholipse C, tyrosine kinase activity, and stress-activated protein kinase p38 are required for this TGF-beta1 effect. Trimeric G proteins and mitogen-activated protein kinases erk1 and erk2 do not appear to be involved. While these results emphasize the complexities involved in the control of extracellular matrix synthesis by TGF-beta, they also identify reaction sites that may be amenable to pharmacologic modulation. Such modulation could be of great advantage in the treatment of a wide variety of undesirable fibrotic reactions