Leesa M. Barone

Bio: Leesa M. Barone is an academic researcher from University of Massachusetts Amherst. The author has contributed to research in topics: Osteoblast & Gene expression. The author has an hindex of 8, co-authored 10 publications receiving 3405 citations.

Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix

Abstract: The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods–;proliferation, extracellular matrix maturation, and mineralization–;and 2) two restriction points to which the cells can progress but cannot pass without further signal–;the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle-and cell growth-regulated genes, produce a fibronectin/type I collagen extracel-lular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phos-phatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Progressive Development of the Rat Osteoblast Phenotype In Vitro: Reciprocal Relationships in Expression of Genes Associated With Osteoblast Proliferation and Differentiation During Formation of the

Abstract: The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periodsproliferation, extracellular matrix maturation, and mineralization-and 2) two restriction points to which the cells can progress but cannot pass without further signals-the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycleand cell growth-regulated genes, produce a fibronectinhype I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Pleiotropic Effects of Vitamin D on Osteoblast Gene Expression Are Related to the Proliferative and Differentiated State of the Bone Cell Phenotype: Dependency upon Basal Levels of Gene Expression, Duration of Exposure, and Bone Matrix Competency in Normal Rat Osteoblast Cultures

Abstract: Normal rat osteoblasts in culture undergo a developmental sequence consisting of a proliferation period in which high levels of the histone and collagen type I genes are expressed, followed by periods of matrix maturation [high levels of alkaline phosphatase (AP)] and mineralization that signal a high level of production of osteopontin (OP) and osteocalcin (OC). Since these parameters are regulated by vitamin D, the effects of both short term and chronic treatment with 1,25- dihydroxyvitamin D3 were examined during osteoblast growth and differentiation. In acute studies, during the proliferation period, histone mRNA (reflecting DNA synthesis) was inhibited (20–60%). Matrix Gla protein (MGP) and OP mRNA were significantly elevated during proliferation (30- and 15-fold), in contrast to OC which is not expressed and was not induced by hormone treatment. OP and MGP remained stimulated throughout the developmental sequence, but to a lesser degree (from 6- to 10-fold). Collagen and AP mRNA were inhibited by hor...

Developmental expression and hormonal regulation of the rat matrix GLA protein (MGP) gene in chondrogenesis and osteogenesis

Abstract: Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed:colon; an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25-(OH)2D3 (10−8 M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post-transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintance of a collagenous matrix as reflected by increases in MGP mRNA during these periods. Moreover, our data suggest that cartilage and bone MGP mRNA may in part be selectively regulated by 1,25-(OH)2D3 at the post-transcriptional level.

Gene expression during endochondral bone development: evidence for coordinate expression of transforming growth factor beta 1 and collagen type I.

Abstract: Subcutaneous implatation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro- and osteo-inductive events which are directly comparable to those which occur in normal endochondral bone development. Although the morphological and biochemical sequence associated with endochondral bone formation in this model has been well characterized, to date little information is available as to the gene regulation by which these events occur. To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell-growth-regulated and tissue-specific genes were examined by slot blot analysis and compared to the morphological changes occuring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c-myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to (1) production of fibroprogenitor cells (day 3), (2) onset of bone formation (day 9), and (3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post-implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7–10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor β1 (TGFβ) were compared to those of collagen types I and II; a direct temporal correlation of TGFβ mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGFβ and type I collagen mRNA levels is consistent with a functional role for TGFβ in extracellular matrix production during in vivo bone formation.

Human Adipose Tissue Is a Source of Multipotent Stem Cells

Abstract: Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Molecular mechanism mediating proliferation, defferentiation interralationships during progressie development of the osteoblast phenotype

Abstract: I. Introduction A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...

Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype

Abstract: I. Introduction A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...

Distinct proliferative and differentiated stages of murine MC3T3‐E1 cells in culture: An in vitro model of osteoblast development

Abstract: We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.