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M J Somerman

Publications and source records attributed to M J Somerman.

At least 55 records · Page 3Linked to original sources
PubMed1998

Isolation of murine cementoblasts: unique cells or uniquely-positioned osteoblasts?

While cementoblasts express a number of mineral-related proteins, including bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), these proteins do not appear to be expressed by cells of the intermediate dental follicle/periodontal ligament (PDL). This information was utilized in an experimental strategy to isolate presumptive cementoblasts from the root surface of day 24 murine mandibular first molars. Using microscopic dissection techniques, molars were carefully extracted from their alveolar crypts and subjected to trypsin-collagenase digestion to remove adherent cells. Primary cultures were established and assayed for expression of proteins known to be expressed by cementoblasts at this timepoint in vivo (i.e. BSP, OPN, OC) and also an odontoblast-specific protein (i.e. DSP) to rule out contamination by pulpal cells. A subgroup of cells were found to express Type I collagen (89% of cells), BSP (46%), OPN (23%) and OC (30%); DSP was not detected within these cultures. We propose that cells within this heterogeneous population, which express this profile of osteogenic proteins, represent cementoblasts. The availability of a cementoblast cell line will make possible rigorous and controlled in vitro analysis of these cells and allow for determination of the unique characteristics of these cells not shared with other cells, particularly osteoblasts.

Animals↗
PubMed1998

Protein extracts of dentin affect proliferation and differentiation of osteoprogenitor cells in vitro.

Proteins associated with the mineral phase of dentin are considered to have the potential to alter cell function within the local environment, during development and regeneration of tooth/periodontal tissues. Cells that may be altered include osteoblasts, ameloblasts, periodontal ligament cells, odontoblasts, and cementoblasts. However, specific factors within dentin controlling cell activity have not been elucidated. To investigate further the role of dentin proteins in regulating cell behavior, MC3T3-E1 cells, a mouse osteoprogenitor cell line, were exposed to guanidine/EDTA extracts of dentin (G/E-D) prepared from bovine teeth. Cells, with or without G/E-D (2 to 50 microg/ml), were evaluated for proliferative activity and for mRNA expression of bone-associated genes. Results indicated that G/E-D suppressed cell proliferation and caused striking morphological changes, including the conversion of cuboidal cells into fibroblastic, spindle-shaped cells. Markers of osteoblast differentiation, osteocalcin and bone sialoprotein mRNA were decreased, while osteopontin mRNA was enhanced in cells exposed to G/E-D. Since transforming growth factor beta (TGFbeta1) has been reported to influence cells in a similar fashion, G/E-D were examined for the presence of and concentration of TGFbeta using slot blot analysis and enzyme immunoassay (ELISA), respectively. These analyses demonstrated that G/E-D contained 6.6 ng/mg of TGFbeta1. Next, cells were exposed to G/E-D in conjunction with anti-TGFbeta1,2,3 antibody. When cells were exposed to antibody, G/E-D-mediated changes in morphology and gene expression were blocked. These results suggest that TGFbeta1 and perhaps other factors in dentin can regulate cell behavior and, therefore, can influence development, remodeling, and regeneration of mineralized tissues.

3T3 Cells↗
PubMed1998

Biologic modifiers in periodontal regeneration.

The specific objective of this article was to update the reader on biologic modifiers being used or suggested for use in therapies directed at regenerating periodontal tissues. As indicated from the studies presented here, many of these biologic modifiers have significant influences on cell behavior and show great promise for use in regenerative therapies. As discussed here, however, additional investigations are required both at the molecular level and at the clinical level to improve the predictability of regenerative therapies. With active investigations directed toward understanding the biology of the healing site, including identifying appropriate cells to target, coupled with designing delivery systems that can control release of agents at the local site, establishing the required environment for regeneration of periodontal tissues should be feasible.

Animals↗
PubMed1997

Promotion of HL-60 cell differentiation by 1,25-dihydroxyvitamin D3 regulation of protein kinase C levels and activity.

The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] promotes differentiation of a number of cell types including HL-60 promyelocytic leukemia cells. It is now established that protein kinase Cbeta (PKCbeta) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCbeta levels and activation in 1,25-(OH)2D3-mediated differentiation of HL-60 cells. Cell differentiation promoted by 1,25-(OH)2D3 at 48 hr was 39 +/- 3% (mean +/- SEM) nitroblue tetrazolium (NBT) positive and at 72 hr it was 35 +/- 2% NBT positive and 70% CD14 positive. Thus, promotion of cell differentiation by 20 nM 1,25-(OH)2D3 treatment was maximal at 48-72 hr. When PKCbeta levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)2D3 for the initial 6 hr increased PKCbeta levels by 175% but had little effect on cell differentiation (7 +/- 2% NBT positive; 11% CD14 positive). The effect of ionomycin, a calcium ionophore, on PKCbeta levels and cell differentiation also was examined. Alone, 5 microM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 microM ionomycin for 66 hr after a 6-hr pretreatment with 20 nM 1,25-(OH)2D3 resulted in 34 +/- 5% NBT positive cells and 73% CD14 positive cells. Quantitatively, this induction of differentiation was identical to that observed in cultures continuously treated with 1,25-(OH)2D3 (35 +/- 2% NBT positive; 70% CD14 positive). Therefore, ionomycin seemed to replace the requirement for the continuous presence of 1,25-(OH)2D3. Chelerythrine chloride (3 microM), a specific PKC inhibitor, blocked differentiation promoted by 1,25-(OH)2D3 alone (82 +/- 2% inhibition) or in sequence with ionomycin (86 +/- 3% inhibition). Taken together, our data show that the capacity of 1,25-(OH)2D3 to both increase PKCbeta levels and activate PKC is utilized to promote HL-60 cell differentiation. These data further suggest that 1,25-(OH)2D3 has a genomic action to increase PKCbeta levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.

Calcitriol↗
PubMed1997

Stimulation of osteopontin mRNA expression in HL-60 cells is independent of differentiation.

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces HL-60 cells to differentiate along the monocyte/macrophage pathway and stimulates expression of the extracellular adhesion protein osteopontin (OPN). In this study, the mechanism of TPA-mediated OPN mRNA expression and its relationship to differentiation were investigated. The induction of OPN mRNA by TPA was dose dependently inhibited by staurosporine (0.4-10.0 nM) and chelerythrine (0.1-5.0 microM), indicating that OPN expression requires PKC activation. Furthermore, the mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD 098059 (1.0-10.0 microM), inhibited the effect of TPA in a dose-dependent fashion. Cycloheximide (10 microg/ml) ablated the induction of OPN mRNA by TPA. To determine if OPN mRNA expression was associated with a particular differentiational pathway, HL-60 cells were treated with RA, 9-cis-RA, calcitriol, or sodium butyrate. None of these agents stimulated OPN mRNA. Treatment with TPA subsequent to a 120-h pretreatment with retinoic acid (RA), 9-cis-RA, or calcitriol resulted in a potentiation of the induction of OPN mRNA. These results support a role for protein kinase C (PKC) in promoting OPN expression because each of these agents increased PKC levels. An hOPN promoter/reporter construct was responsive to TPA, indicating that this effect is at the level of transcription. Thus, TPA-stimulated transcription of the OPN gene apparently occurs via a PKC/MAPK-dependent mechanism that is independent of that associated with differentiation and is not dependent on the maturational state of these cells.

Alkaloids↗
PubMed1997

Expression of bone associated markers by tooth root lining cells, in situ and in vitro.

Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblasts). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface--cementoblasts--with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen, BSP, OPN, and OCN as determined by in situ hybridization. In contrast, cells within this population did not express dentin sialoprotein, an odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cells, or cementoblasts, in vitro.

Animals↗
PubMed1997

Dentin sialoprotein (DSP) transcripts: developmentally-sustained expression in odontoblasts and transient expression in pre-ameloblasts.

Dentin sialoprotein (DSP), a 53 kDa glycoprotein, is believed to be present exclusively in dentin. Using rat and mouse digoxigenin labeled (DIG)-DSP and 35S-DSP riboprobes, and in situ hybridization techniques, we have studied the presence of DSP mRNA at specific developmental stages of dentinogenesis. In mouse and rat molars and incisors, DSP transcripts were localized in young odontoblasts associated with early stages of predentin formation, as well as in mature odontoblasts, cells with cytoplasmic extensions embedded in the forming dentin. No DSP transcripts were detected in dental pulp, enamel organ, ameloblasts, epithelial root sheath, Meckel's cartilage, alveolar bone or tibia. Furthermore, no DSP mRNA was observed in other soft tissues including heart, lung, kidney, intestine, eye, and muscle. In addition to the intense and prolonged expression by odontoblasts, DSP mRNA was transiently expressed by pre-ameloblasts in both developing molars and incisors. These observations are consistent with the results of previous immunohistochemical studies (1). The transient expression of DSP in pre-ameloblasts across from young odontoblasts suggests an involvement of DSP in epithelial-mesenchymal interactions that are crucial to later stages of tooth development.

Alveolar Process↗
PubMed1997

1,25-Dihydroxyvitamin D3 regulation of cardiac myocyte proliferation and hypertrophy.

We previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits myocyte maturation (T. D. O'Connell, D. A. Giacherio, A. K. Jarvis, and R. U. Simpson. Endocrinology 136: 482-488, 1995). To define further the role of 1,25(OH)2D3 in regulating myocardial development, we examined the effects of 1,25(OH)2D3 on proliferation and growth of primary cultures of ventricular myocytes isolated from neonatal rat hearts. When neonatal myocytes were grown in a serum-supplemented medium, cell number approximately doubled, and treating these myocytes with 1,25(OH)2D3 inhibited their proliferation by 56.56% after 4 days. Flow cytometry revealed that 1,25(OH)2D3 reduced the percentage of cells in the S phase of the cell cycle by 31.39% after 4 days. We show for the first time that proliferating cell nuclear antigen protein levels were specifically reduced by 1,25(OH)2D3. Protooncogene c-myc protein levels were also reduced by this hormone. Interestingly, a phorbol ester had a similar effect on myocyte proliferation. Furthermore, 1,25(OH)2D3 increased myocyte protein levels and increased cell size, suggesting that it induces cardiac myocyte hypertrophy. Our findings indicate that 1,25(OH)2D3 and phorbol esters directly regulate myocyte proliferation and induce myocyte hypertrophy. Finally, the data demonstrate that the mechanism by which 1,25(OH)2D3 regulates myocyte proliferation involves blocking entry into the S phase of the cell cycle.

24,25-Dihydroxyvitamin D 3↗
PubMed1997

Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro.

Periodontal regeneration is a complex process that requires coordinated responses from several cell types within the periodontium. It is generally accepted that the periodontal ligament (PDL) has a heterogeneous cell population, where some of the cells may be capable of differentiating into either cementoblasts or osteoblasts. Thus, it has been hypothesized that PDL cells play a role in promoting periodontal regeneration. However, definitive evidence to support this concept is lacking. Previously, we reported that PDL cells induce biomineralization as determined by Von Kossa histochemistry and transmission electron microscopy. To further determine the osteoblast-like properties of PDL cells, human PDL cells were exposed to dexamethasone (DEX) in order to promote an osteoblast phenotype, and then cell activity monitored during mineral nodule formation in vitro. For mineralization studies, cells were cultured in DMEM containing 10% FBS and a) vehicle only; b) ascorbic acid (50 micrograms/ml) and beta-glycerophosphate (10 mM); or c) ascorbic acid, beta-glycerophosphate and DEX (100 nM) for 30 days. In addition, the effects of DEX on PDL cells in non-mineralizing media were determined. Cells were stained weekly to evaluate mineral-like nodules, using the Von Kossa method. Northern blot analyses for mRNA steady state levels for several bone-associated proteins, i.e., osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (ALP), osteocalcin (OCN), alpha 2(1)(type 1) collagen and osteonectin (ON), were performed. DNA levels were also determined during the 30-day mineralization period. Under phase contrast microscopy, PDL cells in non-mineralizing media treated with DEX exhibited a more spindle-shaped morphology when compared with similar cells not exposed to DEX. Mineralizing conditions were required to induce mineral nodule formation. However, in this situation, mineral induction was independent of DEX; and furthermore, DEX-treated cells did not exhibit a different morphological pattern when compared with non-DEX treated cells. Mineral-like nodules were first seen at day 15, in concert with an increase followed by a decrease in expression of type I collagen and ON mRNA in both DEX-treated and non-treated cultures. Using Northern blot analysis for detection of specific proteins, we found that PDL cells did not express OPN, BSP, OCN, or ALP under any of the conditions used in this study. DEX did not alter DNA content in the cultures during the mineralization period. These results confirm that human periodontal ligament cells can be induced to mineralize in vitro and indicate that dexamethasone does not significantly alter the extent and pattern of mineralization.

Alkaline Phosphatase↗
PubMed1996

Partial cDNA sequencing of mouse dentine sialoprotein and detection of its specific expression by odontoblasts.

Dentine sialoprotein (DSP), a 53-kDa acidic glycoprotein, is expressed by odontoblasts and secreted into the dentine extracellular matrix. Although little is known about its biological function, it might play a part in dentinogenesis. Because DSP has only been shown to occur in rat dentine, it is important to demonstrate its existence in another species. Here, the presence of DSP gene in the mouse genome, and the cloning of a mouse DSP cDNA coding for about one-fifth of the molecule with a nucleotide sequence similar to that for rat cDNA, are reported. Using in-situ hybridization, DSP mRNA was uniquely detected in mouse odontoblasts.

Animals↗
PubMed1996

Agents with periodontal regenerative potential regulate cell-mediated collagen lattice contraction in vitro.

A variety of pharmaceutical agents has been proposed for use in periodontal therapy to inhibit loss of alveolar bone and to promote regeneration of tissues lost to disease. The purpose of this study was to determine the effects of such agents on periodontal cell-mediated gel contraction, an in vitro process considered representative of wound contraction and remodeling in vivo. Human gingival fibroblasts were cultured in a type I collagen lattice, and contraction was quantified by means of a computer-assisted video imaging system. Cell-gel combinations were prepared with cells both pre-exposed and non-exposed to agents; non-anchored cell-gels were then incubated with agents for various time periods. Agents tested included Demecolcine (an inhibitor of cytoskeletal contraction), growth factors (i.e., TGF-beta 1, PDGF, and IGF-1), and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin, ibuprofen, naproxen, and flurbiprofen). While Demecolcine inhibited gel contraction, TGF-beta 1 (1-20 ng/mL), PDGF (100 ng/ML), IGF-1 (1000 ng/mL), and [PDGF + IGF], all of which have been reported to enhance wound healing in vivo, promoted lattice contraction in this system. In contrast, NSAIDs inhibited cell-gel contraction. Ethanol, used to solubilize two specific NSAIDs, also inhibited cell proliferation and gel contractile ability, even at very low concentrations. These findings indicate that periodontal cells respond differently to various agents in vitro and may be adversely affected by alcohol. Furthermore, the results of this study suggest that the cell-lattice contraction system holds potential as a method for screening agents for positive or negative effects on cell activity.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Expression of adhesion molecules during tooth resorption in feline teeth: a model system for aggressive osteoclastic activity.

Tooth resorption, a common feline dental problem, is often initiated at the cemento-enamel junction and hence is called cat 'neck' lesion. Studies have demonstrated that osteoclasts/odontoclasts are increased and activated at resorption sites, and that areas of resorption are partly repaired by formation of tissues resembling bone, cementum, and possibly dentin. However, the cellular/molecular mechanisms/factors involved in resorption and repair are unknown. In this study of tissues from cats with 'neck' lesions, we used specific antibodies and immunohistochemical analyses to examine adhesion molecules associated with mineralized tissues, bone sialoprotein (BSP) and osteopontin (OPN), and a cell-surface receptor linked with these molecules, alpha v beta 3, for their localization in these lesions. In addition, to determine general cellular activity during repair, we performed in situ hybridization using a type I collagen riboprobe. Results showed OPN localized to resorption fronts and reversal lines, while BSP was localized to reversal lines. However, some osteoclasts and odontoblasts "sat" on mineralized surfaces not associated with OPN. The cell-surface receptor, alpha v beta 3, was localized to surfaces of osteoclasts/odontoclasts. Type I collagen mRNA was expressed where osteoblasts attempted to repair mineralized tissue. In contrast, odontoblasts did not express mRNA for type I collagen. This study suggests that osteoclastic resorption is the predominant activity in 'neck' lesions and that this activity was accompanied, at least in part, by increased concentrations of OPN and an associated integrin, alpha v beta 3, at resorption sites. Lack of collagen expression by odontoblasts indicates that odontoblasts do not play an active role in attempts at repair.

Animals↗
PubMed1995

Localization and expression of osteopontin in mineralized and nonmineralized tissues of the periodontium.

To summarize results from various studies focusing on determining the expression/localization of BSP and OPN during tooth root development, there is general agreement that OPN is expressed/localized to the root surface during cementogenesis and is also seen throughout the PDL region. The expression/localization of OPN to odontoblasts and its role in dentinogenesis is less apparent. Recent studies directed at establishing odontoblast cell lines should help to resolve this conflict. Studies on BSP expression during tooth root formation indicate a very precise expression and localization of this molecule during cementogenesis indicating that this molecule may play an important role in the formation of this mineralized tissue. However, as with OPN, the expression of BSP and its role in dentin formation is not clearly defined.

Animals↗
PubMed1995

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions.

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins ("delRGD" or "RGE") supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.

3T3 Cells↗
PubMed1995

Enhanced expression of alpha v integrin subunit and osteopontin during differentiation of HL-60 cells along the monocytic pathway.

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for alpha v integrin subunit was observed. The alpha v beta 3 integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.

Antigens, Surface↗