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Biomedical subjects

G A Rodan

Publications and source records attributed to G A Rodan.

At least 19 recordsLinked to original sources
PubMed1992

Impaired bone activity in aged rats: alterations at the cellular and molecular levels.

We have used a model of rapid bone induction and resorption in rats initiated by the removal of bone marrow to define age-associated deficits. Here we report the sequential expression of various genes implicated in the formation and removal of bone following marrow ablation. Significant increases in alkaline phosphatase and procollagen alpha 1(I) mRNA were observed by day 5, and of osteocalcin and osteopontin by day 6. At their peak, these mRNA levels were elevated three- to eight-fold and correlated with histological evidence of bone formation. No change in collagen II mRNA was observed, indicating that there was no cartilage phase. Collagenase activity increased 10-fold at day 9 and coincided with the beginning of bone resorption. Actin mRNA, a reference gene marker, remained at constant levels. Comparison of the response between adult (6 mo.) and old (24 mo.) rats showed the same temporal pattern, but a lower expression of bone-related genes in older rats. Histological examination also showed that the bone volume and osteoblast number at day 6 were significantly lower in old rats. Furthermore, the percentage of mineralized bone was greatly reduced in the aged rat. This model system is currently being used to evaluate the effectiveness of interventions to up-regulate the bone activity in senescent rats.

Aging↗
PubMed1992

Introduction to bone biology.

Bone at the tissue level undergoes remodeling: it is continuously being resorbed and rebuilt (or formed). A negative balance between bone resorption and formation, frequently due to excessive resorption, is the basis of many bone diseases. Resorption is carried out by osteoclasts, which are specialized multinucleated cells of hemopoietic origin. Bone resorption takes place at a specialized area of the osteoclast cell membrane called "ruffled border," which comprises a sealed lysosomal compartment where the acidic pH solubilizes the mineral and the proteolytic enzymes digest the matrix. Among the agents that inhibit bone resorption, only calcitonin and bisphosphonate have been shown to act directly on osteoclasts. Other hormones and agents, which modulate bone turnover, probably act on the osteoblasts or cells of the osteoblast lineage. Osteoblasts are bone-forming cells, originating from cells resident in bone committed to the osteoblastic lineage. They synthesize and secrete most of the proteins of the bone matrix, including type I collagen and noncollagenous proteins. They possess high levels of alkaline phosphatase, which participates in mineralization. Proteins, produced by osteoblasts, spill over into the blood and are used as indicators of bone formation. In addition to the matrix-forming ability, cells of the osteoblastic family (osteocytes, lining cells, and maybe other cells) participate in the regulation of bone turnover. They respond to parathyroid hormone, glucocorticoids, vitamin D, sex steroids, insulin, prostaglandins, growth factors, and so on. There are a significant number of cytokines, that are locally produced and may control bone resorption. These include prostaglandins, IL1, TNF alpha, possibily IL6.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids.

We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNA- and ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligand-binding domain of the new protein and the DNA-binding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (100-250 microM).

Amino Acid Sequence↗
PubMed1992

Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1.

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.

Alkaline Phosphatase↗
PubMed1991

Cell density-dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: correlation with cyclic AMP changes.

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.

Actins↗
PubMed1991

Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP.

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.

Actins↗
PubMed1991

Retinoic acid increases zif268 early gene expression in rat preosteoblastic cells.

In this study we demonstrate that retinoic acid (RA) increases the expression of transcription factor zif268 mRNA in primary cultures of fetal rat calvarial cells and in simian virus 40-immortalized clonal rat calvarial preosteoblastic cells (RCT-1), which differentiate in response to RA, but not in the more differentiated RCT-3 and ROS 17/2.8 cells. The increased expression of zif268 mRNA is rapid (maximal within 1 h), transient (returns to basal levels by 3 h), detectable at RA doses of 10(-12)M, and independent of protein synthesis. The relative stimulation of zif268 mRNA by RA was much larger than that of other early genes, including c-fos, c-jun, and junB. The rate of transcription of RA-stimulated RCT-1 cells, estimated by nuclear run-on assays, was elevated, suggesting that RA regulation of zif268 gene transcription was at least in part transcriptional. Moreover, RA stimulated the transcriptional activity of a Zif268CAT (chloramphenicol acetyltransferase) plasmid containing 632 bp of zif268 5' regulatory sequences in RCT-1 cells but not in the more differentiated RCT-3 cells. These in vitro data support the in vivo observations which localize zif268 and RA receptor-gamma transcripts to bone and cartilage during development, suggesting that both RA and zif268 may play a role in osteoblast differentiation.

Animals↗
PubMed1991

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3H]alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 microns away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone. In vitro alendronate bound to bone particles with a Kd of approximately 1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 x 10(-3) fmol/microns 2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.

Alendronate↗
PubMed1991

Functional estrogen receptors in osteoblastic cells demonstrated by transfection with a reporter gene containing an estrogen response element.

Although a small number of estrogen receptors (ER) were visualized in osteoblastic cells, and estradiol (E2) has some effects on osteoblasts in vitro, the direct action of E2 on osteoblasts has not been fully established. To determine the presence of functional ER in osteoblasts, we transfected cells with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene and the estrogen-responsive element (ERE) from the vitellogenin A2 gene. E2-dependent induction of CAT activity was determined 48 h after transient transfection and subsequent treatment with 10-100 nM 17 beta-E2. 17 beta-E2, but not 17 alpha-E2, dihydrotestosterone, or progesterone, induced CAT activity in a dose-dependent manner (up to 6-fold) in rat calvarial fraction-3, RCT-3, PyMS, and UMR-106 cells as well as in the human osteosarcoma cell line SaOS-2/B-10. In contrast, E2 had no effect on the induction of CAT activity in the preosteoblastic cell lines RCT-1 and TRAB-11, in the rat osteosarcoma cell line ROS 17/2.8, and in the fibroblastic cell lines BALB-c/3T3 and NRK. Over-expression of ER using a simian virus-40-based expression vector not only conferred or enhanced E2-dependent induction of CAT in all cell types, but augmented E2-dependent expression of insulin-like growth factor-I and E2-stimulated DNA synthesis in primary calvarial and PyMS osteoblastic cells, respectively. These data show the presence of low levels of functional endogenous ER in some, but not all, osteoblastic cells and suggest that the abundance of ER may be rate limiting in the action of E2 on these cells.

3T3 Cells↗
PubMed1991

Gene expression in osteoblastic cells.

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Adenylate cyclase and ATPase activities in red cell membranes of patients and genetic carriers of Duchenne muscular dystrophy.

Basal adenylate cyclase activity was increased in red cell ghosts from both patients with Duchenne muscular dystrophy and their mothers when the activities were compared to proper age-matched controls. The activity of ATPase measured in the presence of Na+, K+, and Mg2+ was not found to be different in erythrocyte ghosts from Duchenne dystrophic patients, age-matched controls, or the mothers of Duchenne patients, and ouabain inhibited ATPase in ghosts to the same extent in all membrane preparations.

Adenosine Triphosphatases↗
PubMed1979

Adenylate cyclase in muscular dystrophy.

The purpose of this study was to determine whether the previously reported differences in adenylate cyclase activity between the sarcolemma of normal and dystrophic chick muscles are also found in the SR, to search for a possible relationship between the adenylate cyclase changes and the pathophysiology of dystrophy, and to investigate whether the findings can be extended to Duchenne human muscular dystrophy by studying the adenylate cyclase and ATPase activities of erythrocyte ghosts from DMD patients and carriers. Microsomes were separated by standard techniques from the pectoralis muscles of normal and dystrophic ckeckens of various ages. The microsomal yields were significantly larger in dystrophic muscles. Adenylate cyclase activities in dystrophic microsomes were higher than those in matched controls and increased with the progression of the disease. The ratio between the two rose from one at 2 weeks of age to nine at about 9--10 weeks. Kinetic analyses showed that the ks for MgATP2- was about 40 microM (at 3 mM Mg2+ and 0.3 mM Ca2+) both in normal and dystrophic microsomes, that calcium caused umcompetitive inhibition of the enzyme (Ki = 0.2 mM), that the effect of calcium was noncooperative (Hill coefficient, nH = 1), that calcium did not affect the cooperativity for MgATP2-, and that magnesium competitively removed the calcium inhibition and caused additional, cooperative stimulation of the enzymatic activity (ka = 1.5 mM; NH =2). The major difference between normal and dystrophic adenylate cyclase was a higher enzymatic velocity in the latter, suggesting a larger amount of enzyme. We investigated whether altered cAMP levels may effect calcium accumulation. Calcium uptake measured (in the presence of oxalate) at several ages revealed no difference between normal and dystrophic chickens. The extent of calcium binding was also similar, although the kd for Ca2+ was lower in dystrophic microsomes. Binding was enhanced in the presence of exogenous protein kinase, but the responses of normal and dystrophic tissues were similar. We concluded that the elevation of adenylate cyclase in dystrophy was not related to microsomal calcium accumultion. Ivestigation of the localization of microsomal adenylate cyclase supported this view. Separation of calcium-loaded microsomes on a discontinuous sucrose gradient into four fractions demonstrated that adenylate cyclase activity, measured in the presence of Lubrol-PX and EGTA, was inversely related to calcium-accumulating activity. Na+, K+-ATPase comigrated with adenylate cyclase. Highest specific activities were found in the lightest fraction. These observations were confirmed by histochemical studies. The reaction product from adenylate cyclase activity was present predominantly in the terminal cisternae of the SR. In the context of the literature, our findings suggest that the rises in adenylate cyclase and Na+, K+-ATPase in avian dystrophy are compensatory changes, elicited by a defect in ECC at the calcium release step...

Adenylyl Cyclases↗
PubMed1978

DNA synthesis in cartilage cells is stimulated by oscillating electric fields.

External oscillating electric fields (1166 volts per centimeter, 5 hertz) enhanced the incorporation of [3H] thymidine into the DNA of chondrocytes isolated from the proliferative layer of embryonic (16 days) chick epiphysis. Verapamil or tetrodotoxin at 10(-6)M concentrations completely blocked the electric field effect. Tetracaine reduced the incorporation of [3H] thymidine in both control and electrically stimulated cells. The findings support the hypothesis that Na and Ca2 fluxes generated by the electrical perturbation trigger DNA synthesis in these cells.

Animals↗