Cyclosporin A induces proliferation in human gingival fibroblasts via induction of transforming growth factor-beta1.

BACKGROUND: Cyclosporin A (CsA) is a widely used immunosuppressant that causes significant side effects including gingival overgrowth. The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta 1 (TGF-beta1). In this study, we evaluated the effects of CsA and TGF-beta1 on human normal gingival (NG) fibroblast proliferation, and explored a possible autocrine stimulation of TGF-beta1 as a cellular regulator of proliferation induced by CsA in NG fibroblasts. METHODS: NG fibroblast cell lines were incubated with increasing concentrations of CsA or TGF-beta1 and the proliferation index determined by automatic cell counting, BrdU incorporation, PCNA expression, and mitotic potential. To determine the effect of TGF-beta1 on the proliferation rate of NG fibroblasts under CsA treatment, NG fibroblast cultures were simultaneously treated with CsA and antisense oligonucleotides against the translation-start site of the TGF-beta1 mRNA. RESULTS: Treatment of NG fibroblasts with CsA or TGF-beta1 significantly stimulated the cell proliferation in a dose-dependent manner. Furthermore, neutralization of TGF-beta1 production in CsA-treated NG fibroblasts inhibited CsA's effect on NG fibroblast proliferation, demonstrating an autocrine stimulatory effect of TGF-beta1 in CsA-treated NG fibroblast proliferation. CONCLUSION: The results presented here suggest that CsA stimulatory induction of NG fibroblast proliferation is mediated via TGF-beta1 in an autocrine fashion.

Expression of Sec61 alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61 alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61 alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61 alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61 alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61 alpha amounts. It was also demonstrated that Sec61 alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61 alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61 alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.

Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosis.

OBJECTIVE: The objective of this study was to examine the histomorphometric features and evaluate the expression of epidermal growth factor (EGF) and transmembranic receptor (EGFr) and the proliferative potential of epithelial cells from normal and hereditary gingival fibromatosis (HGF) gingival tissues. BACKGROUND: EGF is a multifunctional cytokine with a variety of biological effects including stimulation of cell proliferation by binding to its specific EGFr. METHODS: Immunohistochemistry was performed to measure EGF and EGFr expression and the epithelial cell proliferation was determined by measuring proliferating cell nuclear antigen (PCNA). RESULTS: Histomorphometric evaluation indicated that in HGF the mean height of the epithelial papillae was higher compared to the normal gingiva (NG), whereas mean epithelial area and number of epithelial papillae were quite similar in both groups. The EGF and EGFr positive cells were observed in the basal, spinous and granular cell layers of both normal and HGF tissues, with a gradual reduction from the basal layer. Although the expressions of EGF and EGFr in the control group were significantly higher than those from HGF, in HGF the epithelial papilla tips showed increased number of proliferating cells and elevated expression of EGF and EGFr. There was a correlation between the proliferative potential of epithelial cells and the expression of EGF or EGFr only in the epithelial papilla tips of HGF gingiva. CONCLUSION: Our data suggest that EGF and EGFr in the oral epithelium of HGF gingiva may stimulate epithelial cell proliferation, with the resultant apical migration of the oral epithelium and formation of the slender deep epithelial papillae; however, without hyperplastic alterations.

Expression of Sec61alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61alpha amounts. It was also demonstrated that Sec61alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells

Effect of transforming growth factor-beta1, interleukin-6, and interferon-gamma on the expression of type I collagen, heat shock protein 47, matrix metalloproteinase (MMP)-1 and MMP-2 by fibroblasts from normal gingiva and hereditary gingival fibromatosis.

BACKGROUND: Increased collagen and extracellular matrix deposition within the gingiva is the main characteristic feature of hereditary gingival fibromatosis (HGF). To date, it is not well established if these events are a consequence of alterations in the collagen and other extracellular matrix molecules synthesis or disturbances in the homeostatic equilibrium between synthesis and degradation of extracellular matrix molecules. Cytokines are important regulators of expression of the profibrogenic genes, including type I collagen and its molecular chaperone heat shock protein (Hsp)47 and proteolytic enzymes degrading extracellular matrix such as matrix metalloproteinases-1 and -2 (MMP-1 and MMP-2). METHODS: In this study, we analyzed the expression and production of type I collagen, Hsp47, MMP-1, and MMP-2 in normal gingiva (NG) and HGF fibroblasts, and investigated the effects of transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) on the expression of these genes by NG and HGF fibroblasts. RESULTS: Our results obtained from semi-quantitative reverse transcription-polymerase chain reactions (RT-PCR), Western blots, enzyme-linked immunosorbent assays (ELISA), and enzymographies clearly demonstrated that the expression and production of type I collagen and Hsp47 were significantly higher in fibroblasts from HGF than from NG, whereas MMP-1 and MMP-2 expression and production were lower in fibroblasts from HGF patients. Addition of TGF-beta1 and IL-6, which are produced in greater amounts by HGF fibroblasts, promoted an increase in type I collagen and Hsp47 and a decrease in MMP-1 and MMP-2 expression. IFN-gamma reduced both type I collagen and Hsp47 expression, whereas it had a slight effect on the expression of MMP-1 and MMP-2. CONCLUSION: These patterns of expression and production suggest that enhanced TGF-beta1 and IL-6 production simultaneously increase the synthesis and reduce the proteolytic activities of fibroblasts from patients with HGF, which may favor the accumulation of extracellular matrix observed in patients with this condition.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37 degrees C and 43 degrees C (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV

Expression of Sec61alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61alpha amounts. It was also demonstrated that Sec61alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.

Peptídeos nascentes de pró-colágeno e outras proteínas são transportadas por intermédio da membrana do retículo endoplasmático por um canal de transporte de proteínas chamado de tanslocon. Sec61alfa, uma proteína transmembrânica do translocon, tem sido apontada como essencial para translocação de cadeias nascentes de polipeptídeos para as cisternas do retículo endoplasmático. Entretanto, não se sabe se Sec61alfa é constitutivamente expressa em células de teratocarcinoma produtoras de colágeno. Além disso, a expressão e a utilização de Sec61alfa podem ser dependentes do estágio de diferenciação celular. Células progenitoras pluripotentes obtidas de muitas linhagens de células de teratocarcinoma, incluindo, por exemplo, células F9 e P19, são capazes de diferenciação em resposta a baixas concentrações de ácido retinóico. Essa diferenciação de células tumorais causa perda de sua carcinogenicidade. Para este estudo, células de teratocarcinoma F9 e P19 de camundongos foram cultivadas em meios de cultura tratados com ou sem ácido retinóico. A expressão de Sec61alfa foi determinada pelo método de "reverse transcriptase-polymerase chain reaction" (RT-PCR). Em condições não tratadas, células F9 expressaram quantidades não detectáveis de Sec61alfa. De forma similar, experimentos de RT-PCR demonstraram que a expressão gênica de Sec61alfa é estimulada nas células F9 após tratamento com ácido retinóico por 72 horas. Não foram detectadas diferenças na expressão de Sec61alfa nas células P19 após tratamento com ácido retinóico. Esses dados demonstram que a expressão de Sec61alfa é aumentada em células de teratocarcinoma F9 após diferenciação com ácido retinóico.

Expression of matrix metalloproteinases in cyclosporin-treated gingival fibroblasts is regulated by transforming growth factor (TGF)-beta1 autocrine stimulation.

BACKGROUND: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta1 (TGF-beta1). The aim of this study was to investigate the potential role of TGF-beta1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-beta1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). METHODS: Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-beta1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-beta1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or antisense oligonucleotides (AON). RESULTS: CsA simultaneously stimulated TGF-beta1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-beta1 production as demonstrated by ELISA, whereas TGF-beta1 mRNA expression levels were not significantly modified. The inhibition of TGF-beta1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-beta1 in CsA-treated human gingival fibroblasts. CONCLUSIONS: The data presented here suggest that TGF-beta1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extracellular matrix.

Expression of Matrix Metalloproteinases in Cyclosporin-Treated Gingival Fibroblasts Is Regulated by Transforming Growth Factor (TGF)-ß1 Autocrine Stimulation / x

Background: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-ß1 (TGF-ß1). The aim of this study was to investigate the potential role of TGF-ß1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-ß1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). Methods: Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-ß1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-ß1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or anti-sense oligonucleotides (AON). Results: CsA simultaneously stimulated TGF-ß1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-ß1 production as demonstrated by ELISA, whereas TGF-ß1 mRNA expression levels were not significantly modified. The inhibition of TGF-ß1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-ß1 in CsA-treated human gingival fibroblasts. Conclusions: The data presented here suggest that TGF-ß1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extra-cellular matrix

Testosterone stimulates proliferation and inhibits interleukin-6 production of normal and hereditary gingival fibromatosis fibroblasts.

Hereditary gingival fibromatosis (HGF) is a rare oral condition characterized by a slow and progressive enlargement of the gingiva, involving both the maxilla and mandible. In vitro, HGF fibroblasts demonstrate a proliferative index significantly higher than fibroblasts from normal gingiva (NG). The objective of this study was to determine the effect of dihydrotestosterone on the proliferation of gingival fibroblasts derived from patients with HGF (n = 4) and from four healthy individuals. Additionally, we analyzed the effect of dihydrotestosterone on interleukin-6 (IL-6) production and determined the expression levels of androgen receptors in NG and HGF fibroblasts. Gingival fibroblasts from NG and HGF were incubated with increasing concentrations of dihydrotestosterone with or without androgen blockers, and cultured for 24 h, and the proliferation index was determined by automated cell counter. IL-6 production, in this system, was quantified using a "capture" enzyme-linked immunosorbent assay (ELISA). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to measure the mRNA expression of androgen receptors. The results indicated that dihydrotestosterone simultaneously downregulates the production of IL-6 and upregulates the cell proliferation. Finasteride and cyprosterone acetate, two anti-androgens, partially reversed these effects. Androgen receptor mRNA expression was identified in both NG and HGF fibroblasts; however, the levels in NG were higher than those observed in HGF. These results show that testosterone coordinates the proliferation and production of IL-6 of normal and HGF fibroblasts.

Neurofibromatosis type 1: a clinicopathological study of the orofacial manifestations in 6 pediatric patients.

Neurofibromatosis type 1 (NF1) is a relatively frequent mucocutaneous syndrome, which is transmitted as an autosomal dominant trait or which may represent neomutation. It is characterized by a variety of clinical manifestations, including multiple neurofibromas that are associated with a high risk of sarcomatous transformation. The aim of this report was to elucidate the orofacial manifestations observed in 6 pediatric patients (between 4 and 15 years of age) diagnosed with NF1. Physical, clinical, radiological, histological, and immunohistochemical studies were performed. Orofacial lesions were observed in all studied patients, located either in the soft tissues (4 cases) or centrally in the jaws (2 cases). All cases showed facial asymmetry, one of them exhibiting marked facial hemihypertrophy. All cases with soft tissue involvement were plexiform neurofibromas, while the intraosseous cases were diagnosed as solitary neurofibromas. Knowledge of the variability of presentation of orofacial soft tissue and bone manifestations of NF1 in children is necessary for prompt diagnosis.

Amplification and protein expression of chromosome 12q13-15 genes in osteosarcomas of the jaws.

Overexpression and amplification of several genes (MDM2, CDK4 and SAS) located on chromosome 12q13-15 have been noted to occur in various human sarcomas. As a result, two major growth regulation pathways may be inhibited. MDM2 may down regulate the p53-mediated growth control and CDK4 may affect pRB-mediated events. To determine the frequency of alterations in these genes and their correlation with clinicopathologic features, we analyzed the MDM2 and CDK4 protein levels by immunohistochemistry and assessed MDM2, CDK4 and SAS amplification by real-time PCR in nine osteosarcomas of the jaws. Positive staining for CDK4 and MDM2 was observed in eight cases (88.8%) and five cases (55.5%), respectively. Intense CDK4 staining was noted in four cases (two high grade, one intermediate grade and one low grade). Intense MDM2 staining was observed in the same four previous cases, as well as, one additional high-grade tumor. Individual DNA amplification for CDK4, MDM2 and SAS was observed in six cases for each gene. Co-amplification was observed in five cases that showed CDK4 and MDM2 concomitant amplification and four cases that displayed amplification for all of the genes. In addition, among the five cases that presented CDK4 and MDM2 amplification, strong overexpression of CDK4 and MDM2 was observed in three and in four cases, respectively (three high grade and one intermediate grade). These results suggest that 12q13-15 genes are involved in neoplastic disease and concurrent amplification and overexpression of these genes might help to define high-grade tumors.

Transforming growth factor-beta1 autocrine stimulation regulates fibroblast proliferation in hereditary gingival fibromatosis.

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a rare oral disease characterized by a slow and progressive enlargement of both the maxilla and mandible gingiva. Increased proliferation, elevated synthesis of extracellular matrix, particularly collagen, and reduced levels of matrix metalloproteinases seem to contribute to the pathogenesis of gingival overgrowth in HGF patients. Transforming growth factor-beta1 (TGF-beta1) is an important cytokine thought to play a major role in fibrotic disorders such as HGF due to its ability to stimulate the synthesis and reduce the degradation of extracellular matrix. In HGF fibroblasts, TGF-beta1 autocrine stimulation reduces expression and production of matrix metalloproteinases. However, the role of TGF-beta1 in fibroblast growth modulation has not been established in this disease. METHODS: The aim of this study was to confirm the increased proliferation rate of HGF fibroblast cell lines and to explore a possible autocrine role of TGF-beta1 as a cell growth stimulator by blocking production of this endogenous cytokine using 2 well-established systems: antisense oligonucleotides and neutralizing antibodies. RESULTS: Four different cellular proliferation assays, bromodeoxyuridine labeling, argyrophilic nucleolar organizing region staining, proliferating cell nuclear antigen, and mitotic indexes, confirmed that fibroblasts from HGF proliferate significantly faster than those from normal gingiva. Antisense oligonucleotides reduced TGF-beta1 production as demonstrated by capture enzyme-linked immunosorbent assay, whereas TGF-beta1 expression levels were not significantly modified. Blocking TGF-beta1 synthesis with oligonucleotides or its activity with specific antibodies resulted in a decreased magnitude of HGF fibroblast proliferation. CONCLUSION: These results are consistent with the existence of an autocrine role of TGF-beta1 as a stimulator of HGF fibroblast proliferation.

Increase in expression of Hsp47 and collagen in hereditary gingival fibromatosis is modulated by stress and terminal procollagen N-propeptides.

HGF is a rare oral condition characterized by a slow, progressive enlargement of the gingiva, involving both the maxilla and mandible. HGF provides a model for the study of regulatory features of conditions characterized by connective tissue hyperplasia. In this study, the culture characteristics of gingival fibroblasts derived from patients of the same family with HGF (n = 4) were similar with regard to cell cycle analysis. Flow cytometric DNA content analysis revealed uniform DNA diploidy for fibroblasts cultured from NG and HGF. NG cells showed a low S-phase fraction (19.8%) and G2/M fraction (5.8%) and a relatively high G1 phase fraction (74%). In contrast, HGF cells from all members of the tested kindred, exhibited diploid cells with a higher S-phase (40.9%) and G2/M (10.1%) fraction and a relatively low G1 phase fraction (40.9%). Furthermore, we demonstrated that the expression and production of Hsp47 parallels the increased levels of collagen secretion observed in HGF. In addition, we show that Hsp47 and collagen are coordinately regulated following stress via a feedback mechanism mediated by N-terminal procollagen propeptides. Utilizing confocal microscopy and antibodies directed against GST-fusion proteins encompassing the pro alpha1(I) N-propeptide globular domain (NP1) (residues 23-108), it was apparent that this regulatory mechanism does not involve significant interaction with Hsp47's chaperoning of procollagen.

Alteration in expression of MMP-1 and MMP-2 but not TIMP-1 and TIMP-2 in hereditary gingival fibromatosis is mediated by TGF-beta 1 autocrine stimulation.

Hereditary gingival fibromatosis (HGF) is characterized by an excess accumulation of extracellular matrix (ECM) resulting in a generalized and fibrotic enlargement of the gingiva. To investigate some of the regulatory features of this condition, gingival fibroblasts from normal gingiva (NG) and HGF were examined for the expression and production of matrix metalloproteinases (MMPs) and their inhibitors, tissue matrix metalloproteinases inhibitor (TIMPs). Our results, obtained from 2 different assays, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography, clearly demonstrated that the expression and production of MMP-1 and MMP-2 was significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher to those from HGF. Addition of antibodies against transforming growth factor-beta 1 (TGF-beta 1), which is produced in greater amounts by HGF fibroblasts, resulted in a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas TIMP-1 and TIMP-2 expressions were unaffected. These patterns of expression and production suggest that enhanced TGF-beta 1 production reduce the proteolytic activities of HGF fibroblasts, which favor the accumulation of ECM.

The acro-osteolysis syndrome: Morphologic and biochemical studies.

The acro-osteolysis syndrome consists of dissolution of terminal phalanges of the hands and feet, dolichocephaly with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, a prominent occipital ridge, skeletal demineralization, vertebral and extremity fractures, joint laxity, and coarse hair. Studies of bone morphology reveal diminished bone density and bone formation. Osteoblasts have widely dilated smooth endoplasmic reticulum. It is postulated that an abnormality of a structural protein is the pathogenic basis of this disease.