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Biomedical subjects

M Centrella

Publications and source records attributed to M Centrella.

At least 91 records · Page 5Linked to original sources
PubMed1989

The role of growth factors in skeletal remodeling.

Bone remodeling is a complex process regulated by systemic agents and local factors. During the past several years most or perhaps all the growth factors associated with the bone matrix have been characterized, and important information regarding hematologic factors has become available. Growth factors have significant effects on bone remodeling and likely play a major role in the maintenance of normal bone mass, wound healing, and fracture repair (Tables 3 and 4). Current studies indicate that systemic hormones regulate the synthesis and receptor binding of a number of local factors. By these mechanisms, specific hormones may target their effects to the skeleton. Although our knowledge of local growth factors is still limited, future work should determine their role in skeletal physiologic and pathophysiologic conditions and their potential as therapeutic agents.

Animals↗
PubMed1988

Further biochemical and molecular characterization of primary rat parietal bone cell cultures.

Primary bone cell cultures are used widely to examine the regulation of bone metabolism by growth factors and hormones. Characterization of this model system is now being conducted at the molecular level to define modulation of gene expression. Cells were obtained from rat parietal bone by sequential collagenase digestions. Cell populations were evaluated for bone-related products, including collagen isoform expression and mRNA levels, alkaline phosphatase activity, and osteocalcin production. Serum-deprived, confluent cultures of the first and second collagenase-released populations produced a lower percentage of total protein as collagen than the third, fourth, and fifth populations, while co-culturing the third through fifth populations resulted in the highest level. Collagen typing on SDS-polyacrylamide gels revealed an abundance of mature type I collagen in all cell populations; type III collagen synthesis was undetectable by this method. This is in contrast to the presence of cytoplasmic mRNA for both type I and type III collagen in all cell populations, suggesting post-transcriptional modulation of type III collagen synthesis. The expression of alkaline phosphatase and osteocalcin was highest in cultures of later released cells, indicating that these cell populations display phenotypic characteristics associated with cells of the osteoblast lineage.

Alkaline Phosphatase↗
PubMed1988

Isolation of growth factors from adult bovine bone.

Fetal rat calvariae synthesize transforming growth factor beta (TGF beta), beta 2 microglobulin (beta 2 m), and insulinlike growth factor I (IGF I), but, except for TGF beta, it is not known if these polypeptides are also present in adult bone tissue. Pulverized bovine bone, extracted with 0.5 N HCl and 4 M guanidine HCl and fractionated by gel filtration, was found to contain several biologically active components when tested for its effects on DNA synthesis in osteoblast-rich cell cultures. TGF beta, beta 2 m, and IGF I were identified and further purified using high performance liquid chromatography (HPLC). TGF beta, identified by a standard TGF beta bioassay or by immunoreactivity, was purified by muBondapak C18 and muBondapak CN reversed phase HPLC. beta 2 m, identified by immunoreactivity, required an additional fractionation step on a DEAE-HPLC column for complete purification. IGF I, identified by immunoreactivity, was purified by HPLC using a muBondapak C18 and a DEAE-HPLC column. Purified TGF beta, beta 2 m, and IGF I migrated as single bands on polyacrylamide gel electrophoresis with respective molecular masses of 24,000, 10,000, and 7,500. In conclusion, adult bone matrix, like fetal bone cultures, contains TGF beta, beta 2 m, and IGF I and these factors may play a role in adult skeletal remodeling.

Animals↗
PubMed1988

Adenylate cyclase-stimulating, bone-resorbing and B TGF-like activities in canine apocrine cell adenocarcinoma of the anal sac.

Canine apocrine cell adenocarcinoma of the anal sac (APO-AS) is a spontaneously occurring tumor that causes humorally mediated hypercalcemia in 90% of cases. To further define the nature of the responsible mediator in APO-AS, we examined tumor extracts from five APO-AS and four control tumors for adenylate cyclase-stimulating activity (ACSA). All extracts from APO-AS contained potent ACSA, whereas the four control tumors did not. The ACSA extracted from one tumor demonstrated a dose response curve parallel to that of synthetic bovinePTH-(1-34) and was 80% inhibited by Nle8,18,Tyr34 bPTH-(3-34)amide at a concentration of 10(-5) M. Extracts from three APO-AS and three control tumors were also examined for in vitro bone-resorbing activity (BRA). All APO-AS contained significant BRA, stimulating resorption 1.47 to 2.13-fold over basal, whereas none of the control tumors stimulated resorption. Purification of one extract using C18 reverse-phase high pressure liquid chromatography (RP-HPLC) resulted in a single sharp peak of ACSA which was 400-fold purified compared with the initial extract. This pool also contained significant bone-resorbing activity, whereas none of the adjacent pools did. Purification of a second extract using sequential CN and C18 RP-HPLC followed by size exclusion HPLC resulted in material that was at least 10,000-fold purified, and showed co-purification of ACSA and B TGF-like activity.

Adenocarcinoma↗
PubMed1988

Parathyroid hormone modulates transforming growth factor beta activity and binding in osteoblast-enriched cell cultures from fetal rat parietal bone.

Transforming growth factor beta (TGF-beta) is produced by bone cells, is abundant in bone matrix, and regulates bone cell biochemical processes. In osteoblast-enriched fetal rat parietal bone cell cultures, low TGF-beta doses increase DNA synthesis, whereas higher levels are less mitogenic, stimulate collagen production, and decrease alkaline phosphatase activity. Parathyroid hormone by itself has minimal effects on these processes, but it opposes the effects of TGF-beta and alters TGF-beta binding to its receptors in osteoblast-enriched cultures. Some functions ascribed to parathyroid hormone in bone may therefore result from alterations in TGF-beta activity, suggesting that the local effects of TGF-beta in bone are under systemic hormonal control.

Animals↗
PubMed1988

Skeletal tissue and transforming growth factor beta.

Normal skeletal growth results from a balance between the processes of bone matrix synthesis and resorption. These activities are regulated by both systemic and local factors. Bone turnover is dynamic, and skeletal growth must be maintained throughout life. Although many growth promoters are associated with bone matrix, it is enriched particularly with transforming growth factor beta (TGF-beta) activity. Experimental evidence indicates that TGF-beta regulates replication and differentiation of mesenchymal precursor cells, chondrocytes, osteoblasts, and osteoclasts. Recent studies further suggest that TGF-beta activity in skeletal tissue may be controlled at multiple levels by other local and systemic agents. Consequently, the intricate mechanisms by which TGF-beta regulates bone formation are likely to be fundamental to understanding the processes of skeletal growth during development, maintenance of bone mass in adult life, and healing subsequent to bone fracture.

Alkaline Phosphatase↗
PubMed1988

Effects of basic fibroblast growth factor on bone formation in vitro.

Basic fibroblast growth factor (bFGF) was studied for its effects on bone formation in cultured rat calvariae. bFGF at 0.1-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to 4.4-fold. bFGF also increased the number of colcemid-induced metaphase arrested cells and the DNA content. Transient (24 h) treatment with bFGF enhanced [3H]-proline incorporation into collagen 24-48 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. The collagen stimulated by bFGF was type I, and this effect was observed primarily in the periosteum-free bone. In contrast, continuous treatment with bFGF for 24-96 h inhibited [3H]proline incorporation into type I collagen. bFGF did not alter collagen degradation. In conclusion, bFGF stimulates calvarial DNA synthesis, which causes an increased number of collagen-synthesizing cells, but bFGF has a direct inhibitory effect on collagen synthesis.

Animals↗
PubMed1988

Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae.

Cultured bones have been shown to secrete local regulators of bone remodeling, such as beta 2-microglobulin, transforming growth factor-beta, and insulin-like growth factor (IGF), but the IGF secreted has not been characterized. In the present study, IGF from medium conditioned by 21-day-old fetal rat calvariae was isolated and characterized. IGF was purified using dialysis, gel filtration, and reverse phase HPLC. Amino acid composition was compatible with that of IGF I (somatomedin-C), and amino-terminal sequence analysis revealed homology with IGF-I. The concentration of IGF-I in the calvarial culture medium was 1 nM and was suppressed by cycloheximide. Calvaria-derived rat IGF I at 20 nM stimulated DNA and collagen synthesis by 42% and 26%, respectively, in monolayer cultures of osteoblast-rich rat parietal bone cells. This study indicates that locally produced IGF-I regulates bone formation in cultures of 21-day-old fetal rat calvariae.

Amino Acid Sequence↗
PubMed1988

Insulin-like growth factor I has independent effects on bone matrix formation and cell replication.

The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3-3H]proline and [methyl-3H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [3H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [3H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

Animals↗
PubMed1988

Tumor necrosis factor-alpha inhibits collagen synthesis and alkaline phosphatase activity independently of its effect on deoxyribonucleic acid synthesis in osteoblast-enriched bone cell cultures.

Tumor necrosis factor-alpha (TNF alpha), a product of activated monocytes, induces tissue wasting in certain solid tumors in vivo and in in vitro model systems. Recent studies indicate that TNF alpha also regulates cell replication and expression of differentiated function in a variety of nonneoplastic cell systems. Since monocyte products could accumulate in bone with trauma, inflammation, or other disease states, bone cell activity might be altered by the presence of these pathophysiological molecules. Using cells obtained by sequential enzyme release from fetal rat parietal bone, we find that TNF alpha has acute stimulatory and inhibitory effects on bone cell macromolecular synthesis. Within 24 h of exposure, recombinant human TNF alpha at 0.3-100 nM progressively increases the rate of DNA synthesis in osteoblast-enriched cell cultures up to 3- to 4-fold, and 3-100 nM TNF alpha reduces collagen production and alkaline phosphatase activity by 20-30%. These decreases are not altered by 1 mM hydroxyurea, which blocks the mitogenic effect of TNF alpha by 85-90%. In addition, hydroxyproline levels in the culture medium do not increase relative to the control value after TNF alpha treatment, suggesting that decreased collagen production results from less synthesis rather than increased collagen degradation. Hybridization studies with cDNA encoding the alpha 1-chain of rat type I collagen show that TNF alpha increases type I collagen mRNA to an extent similar to its effect on cell replication. Therefore, TNF alpha appears to inhibit collagen synthesis and alkaline phosphatase activity in osteoblast-enriched cell cultures by mechanisms that are not related to its effects on cell replication.

Alkaline Phosphatase↗
PubMed1988

Interleukin-1 in combination with transforming growth factor-alpha produces enhanced bone resorption in vitro.

Interleukin-1 (IL-1) and transforming growth factor-alpha (TGF alpha) both stimulate bone resorption. We examined the effects that the combination of these two agents had on fetal rat long bone cultures. The 48-h resorptive response to recombinant human TGF alpha was markedly enhanced in the presence of IL-1 (either purified from stimulated human monocytes or recombinant human IL-1 alpha) compared to the effects of either agent alone. The enhanced resorptive response to the combination appeared to be dependent on prostaglandin (PG) synthesis, since it was associated with an increase in PGE concentrations in the medium and was completely blocked by either indomethacin or flufenamic acid. Substitution of TGF alpha with epidermal growth factor, a TGF alpha analog, produced identical results. We also found that IL-1 inhibited the mitogenic response of the cultures to TGF alpha. The effects of IL-1 and TGF alpha on PGE concentrations in the medium and DNA synthesis were similar in the osteoblast-like cell line MC3T3-E1. Activated macrophages and certain malignant cells are capable of producing both IL-1 and TGF alpha. Hence, similar interactions could occur in vivo and may regulate some of the effects that either immune or malignant cells have on bone.

Animals↗
PubMed1987

Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone.

Transforming growth factor beta (TGF beta) stimulates cell replication in fetal rat calvariae, and studies with isolated bone cells suggest that the primary mitogenically responsive cell is of the osteoblast lineage. The effect of TGF beta on bone cell replication is biphasic and depends on both the TGF beta concentration and cell density in monolayer culture. After 23 h of treatment, DNA synthesis in confluent cells is progressively enhanced by 0.15-15 ng/ml TGF beta; but in subconfluent cells, 15 ng/ml is less than maximal; and in sparse cell cultures, it is inhibitory. At all cell densities, however, 15 ng/ml TGF beta stimulates collagen synthesis, an effect which is more pronounced when DNA synthesis rates are declining. Furthermore, 1 mM hydroxyurea, which blocks the mitogenic effect of TGF beta by 85%, only minimally influences the increase in collagen synthesis. Cytoplasmic slot blot analysis reveals alterations in the amount of type I collagen mRNA in TGF beta-treated cells, suggesting that control is exerted, at least in part, at the transcriptional level. Since TGF beta is found in culture medium conditioned by bone explants and in bone tissue extracts, these results support that TGF beta is an important and multifunctional autocrine regulator of bone formation.

Animals↗
PubMed1987

Isolation of EGF-dependent transforming growth factor (TGF beta-like) activity from culture medium conditioned by fetal rat calvariae.

A transforming growth factor of the beta class (TGF-beta), defined by its ability to induce normal rat kidney cells (NRK, clone 49F) to form anchorage-independent large colonies in soft agar in the obligate presence of epidermal growth factor, has been prepared from culture medium conditioned by fetal rat calvariae. This activity was purified by acetic acid extraction, gel permeation chromatography, and two reversed-phase HPLC (rpHPLC) steps. Bone culture derived-TGF beta-like activity was soluble in 1.0 M acetic acid, eluted from Sephadex G-75 at relative molecular mass (Mr) 25,000, from mu Bondapak C18 rpHPLC at 63 +/- 5% methanol in 0.1 M acetic acid, and from mu Bondapak CN rpHPLC at 36 +/- 2% n-propanol in 0.1% trifluoroacetic acid. Based on specific activity estimations at each stage of purification, TGF beta-like activity was purified 2500-fold with a 14% recovery, and 1 l of conditioned medium yielded 1-2 micrograms of factor. Silver-stained polyacrylamide gels of this material after CN mu Bondapak rpHPLC revealed a predominant band of (Mr) 24,000.

Animals↗
PubMed1987

Mitogenesis in fetal rat bone cells simultaneously exposed to type beta transforming growth factor and other growth regulators.

Type beta transforming growth factor (TGF-beta) is found in large amounts in bone tissue, and is a potent mitogen for osteoblast-enriched cell cultures obtained from fetal rat parietal bone. Because other local and systemic factors may be presented to bone cells simultaneously with TGF-beta, it is important to understand the effects of this complex growth regulator in such circumstances. Unlike the effects observed in many tissue systems, TGF-beta does not invariably inhibit the mitogenic response of bone cells to other growth promoters. In contrast, other factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and type alpha tumor necrosis factor (TNF-alpha) limit the response of osteoblastic bone cells to TGF-beta. TGF-beta is a much weaker mitogen for fibroblastic cells obtained from fetal rat bone, whereas fetal bovine serum, EGF, bFGF, and TNF-alpha are more potent stimulators. In addition, TGF-beta does not significantly impair the response of the fibroblastic bone cells to the other tested agents. These findings reinforce a role of TGF-beta as an anabolic bone growth regulator, and suggest that its function may be modified by other local or systemic agents that can also affect bone cells.

Animals↗
PubMed1987

Co-purification of transforming growth factor beta-like activity with PTH-like and bone-resorbing activities from a tumor associated with humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy (HHM) is caused by a circulating bone-resorbing factor or factors. Suggestions as to the nature of this factor include PTH-like proteins, transforming growth factors, and bone-resorbing factors distinct from either of the first two classes of polypeptides. We investigated the occurrence of these three activities in a highly purified extract of the H-500 Leydig cell tumor which causes HHM when implanted into Fisher rats. PTH-like adenylate cyclase-stimulating activity (ACSA) was extracted from tumor tissue by sequential treatment with urea/HCl and ethanol/NaCl. Tumor extract was further purified by hydrophobic-interaction, gel-filtration, and reverse-phase HPLC steps to a specific activity of 1038 ng eq bPTH(1-34)/mg protein. Only the fraction pool containing ACSA demonstrated significant bone-resorbing (1.78-fold over basal) and transforming growth factor activity (epidermal growth factor (EGF)-dependent colony formation in soft agar suspension by NRK-49F indicator cells). A subsequent reverse-phase HPLC step produced material which contained both ACSA and transforming growth factor beta (TGF beta)-like activity in a single fraction. Whether the responsible mediator in this animal model has TGF beta-like properties as well as PTH-like and bone-resorbing activity remains to be determined.

Adenylyl Cyclases↗
PubMed1987

A bone-derived growth factor isolated from rat calvariae is beta 2 microglobulin.

Rat calvariae are known to secrete a bone-derived growth factor (BDGF) that stimulates bone DNA and collagen synthesis. BDGF was purified from calvarial culture medium with dialysis, gel filtration and HPLC. Amino acid composition of BDGF was compatible with murine beta 2 microglobulin (beta 2 m), and amino terminal sequence analysis revealed identity with mature murine beta 2 m. This was confirmed by Western blot analysis using a polyclonal antibody to beta 2 m. Like BDGF, human beta 2 m stimulated bone DNA, collagen and noncollagen protein synthesis. Thus, BDGF, an autologous regulator of bone formation, is homologous to beta 2 m.

Amino Acid Sequence↗