Donald A. Young

Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair

Preclinical and clinical studies suggest a possible role for cyclooxygenases in bone repair and create concerns about the use of nonsteroidal antiinflammatory drugs in patients with skeletal injury. We utilized wild-type, COX-1(-/-), and COX-2(-/-) mice to demonstrate that COX-2 plays an essential role in both endochondral and intramembranous bone formation during skeletal repair. The healing of stabilized tibia fractures was significantly delayed in COX-2(-/-) mice compared with COX-1(-/-) and wild-type controls. The histology was characterized by a persistence of undifferentiated mesenchyme and a marked reduction in osteoblastogenesis that resulted in a high incidence of fibrous nonunion in the COX-2(-/-) mice. Similarly, intramembranous bone formation on the calvaria was reduced 60% in COX-2(-/-) mice following in vivo injection of FGF-1 compared with either COX-1(-/-) or wild-type mice. To elucidate the mechanism involved in reduced bone formation, osteoblastogenesis was studied in bone marrow stromal cell cultures obtained from COX-2(-/-) and wild-type mice. Bone nodule formation was reduced 50% in COX-2(-/-) mice. The defect in osteogenesis was completely rescued by addition of prostaglandin E2 (PGE(2)) to the cultures. In the presence of bone morphogenetic protein (BMP-2), bone nodule formation was enhanced to a similar level above that observed with PGE(2) alone in both control and COX-2(-/-) cultures, indicating that BMPs complement COX-2 deficiency and are downstream of prostaglandins. Furthermore, we found that the defect in COX-2(-/-) cultures correlated with significantly reduced levels of cbfa1 and osterix, two genes necessary for bone formation. Addition of PGE(2) rescued this defect, while BMP-2 enhanced cbfa1 and osterix in both COX-2(-/-) and wild-type cultures. Finally, the effects of these agents were additive, indicating that COX-2 is involved in maximal induction of osteogenesis. These results provide a model whereby COX-2 regulates the induction of cbfa1 and osterix to mediate normal skeletal repair.

Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo‐oxygenase 2 (COX‐2) and mice that lack either COX‐1 (COX‐1−/−) or COX‐2 (COX‐2−/−) to show that COX‐2, but not COX‐1, plays an important role in wear debris‐induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild‐type and COX‐1−/− mice. COX‐2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild‐type and COX‐1−/− mice, COX‐2−/− mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX‐2 is an important mediator of wear debris‐induced osteolysis and suggests that COX‐2 inhibitors are potential therapeutic agents for the prevention of wear debris‐induced osteolysis.

In Vitro Studies of Glucocorticoid Effects on Neurons and Astrocytes

Studies using immunocytochemistry and RNase protection assay demonstrate that glucocorticoid and mineralocorticoid receptors (GR, MR) and their corresponding mRNAs are co-expressed in hippocampal neurons cultured in serum-free, defined medium and at lower levels in cultured astrocytes. Addition of serum or medium conditioned by astrocytes increases the levels of MR mRNA, but has little effect on the levels of GR mRNA. Cellular levels of both GR mRNA and MR mRNA are upregulated by growth of embryonic hippocampal neurons in corticosterone. This is in distinct contrast to regulation of receptor expression in vivo where mRNAs for these receptors are downregulated in the rat hippocampus by corticosterone treatment of the adult adrenalectomized rat. However, in cultured astrocytes, GR and MR mRNAs are also downregulated by corticosterone. To begin to define the role of glucocorticoids in gene expression in astrocytes, we have used giant two-dimensional (2D) gel electrophoresis to separate astrocyte cellular proteins and translation products synthesized in vitro from astrocyte poly A+ RNA. Analysis of approximately 1,500 in vitro translation products by giant 2D gel electrophoresis reveals 11 protein inductions and 1 repression that occur at the level of mRNA in the absence of protein synthesis following treatment of astrocytes with corticosterone. Interestingly, these changes appear to be mediated by GR, but not by MR. The in vitro studies described here are relevant to identifying the role of GR and MR in gene expression in specific cell types in the hippocampus.

An In vitro effect of physiological levels of cortisol and related steroids on the structural integrity of the nucleus in rat thymic lymphocytes as measured by resistance to lysis

Abstract The ability of rat thymus cell nuclei to survive lysis of cells by hypotonic shock (dilution into 1.5 mM MgCl2) is decreased by incubation of whole cells with physiological levels of glucocorticoid hormones in vitro. This effect on “nuclear fragility” is first discernible after 0.5–1 h of incubation with hormone as an increase in non-sedimentible DNA in whole cell lysates. It is measureable at 2 h by counting numbers of surviving nuclei, and by changes in the distribution of DNA and protein from lysed cells on sucrose gradients. Evidence is presented to show that the effect is initiated by a series of molecular events characteristically similar to those which initiate the metabolic effects of the hormone, including specific binding of hormone to receptors and events that are sensitive to cycloheximide. The effect differs from effects of the hormone on many transport and biosynthetic processes which occur at the same time (1–3 h) in as much as the nuclear effect does not require the presence of a carbohydrate energy source in order to become apparent. The specificity and time of onset of the decrease in nuclear integrity combined with the fact that it is reflective of changes in nuclear structure, make it quite likely that it is a simple, rapid assay in vitro of the lymphocytolic effects of Cortisol.

Inhibition of ribonuclease Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, proteinase K and heparin using a sensitive ribonuclease assay

The effectiveness of several commonly used inhibitors of ribonuclease (RNAase) has been studied using the removal of radio-labelled leucine from leucyl-tRNA as a sensitive assay for RNAase activity. The inhibitors were tested under a variety of conditions, varying the temperature, the pH, and the source of RNAase. When each inhibitor is udes separately in the presence of pancreatic RNAase, sodium dodecyl sulfate (SDS) is the most effective; but during long exposures to temperatures above 0 degrees C considerable amounts of RNA are still degraded. Combination of inhibitors are more effective in preserving RNA; with this assay, a combination of SDS with diethyl pyrocarbonate is the most effective. Proteinase K acts as an inhibitor when used in combination with SDS; however, it has RNAase activity when used by itself. Diethyl pyrocarbonate, when used at the high range of concentrations employed by others for RNAase inhibition, reacts with RNA changing its charge. However, when diethyl pyrocarbonate is used in smaller amounts the effects on RNA are minimal, and when used in combination with SDS it effectively inhibits RNAase.

An Early Cordycepin-Sensitive Event in the Action of Glucocorticoid Hormones on Rat Thymus Cells in vitro: Evidence that Synthesis of Mew Mrna Initiates the Earliest Metabolic Effects of Steroid Hormones

Evidence is presented that the synthesis of poly A is essential for the progression of the sequence that follows binding of glucocorticoid hormones to nuclear receptor-complex and leads, after 20–30 min, to the well-known hormonal inhibition of glucose transport. Action of dexamethasone or cortisol on glucose is prevented by amounts of Cordycepin (3′deoxyadenosine) that rapidly block, by 40%, the appearance of poly A in polysomal fractions. Sensitivity to Cordycepin is limited to the first 10 min following hormone addition. In dose-response studies a parallelism is found between degrees of blockade of hormone action and blockade of poly A formation.Studies of possible actions of Cordycepin on: binding of hormones to nuclear receptors, levels of ATP, and synthesis of protein, DNA and nuclear non-poly A RNA are all consistent with the interpretation that the antibiotic blocks hormone action either through its selective effects on poly A synthesis or disturbances in RNA metabolism related to reduced poly A s...

The hepatic glucocorticoid domain: evidence for early and late hormone-mediated changes in the synthesis of individual protein gene products

Studies were conducted to determine the extent of rapidly evolving effects of glucocorticoids on the transcriptional activity of individual hepatocyte genes through comparisons of the relative rates of synthesis of the more than 3000 protein gene products that are resolved in giant two-dimensional separatory gels. During the first 20 h in primary culture normal hepatocytes displayed substantial spontaneous changes in over 80 proteins. One effect of an added glucocorticoid, dexamethasone, was to retard or reverse the progression of roughly half of these. However, such long-term hormone treatment also caused 27 inductions and 26 repressions, many of which occurred in proteins that do not change spontaneously. Some of these coincide with the previously reported glucocorticoid domain of hepatoma cells. In contrast to such long-term changes, short-term (4 h) incubation with dexamethasone induced 10 proteins and repressed 6 others. Five of these early hormone inductions and all of the early repressions were maintained or enhanced by 20 h. However, the remaining five early glucocorticoid inductions appeared to be transient, since by 16-20 h the effects were either markedly reduced or absent. These results show the existence of an early glucocorticoid domain, qualitatively different from that seen at later times, which may be more representative of the primary steroid hormone responses.

Two-dimensional gel analysis of secreted proteins induced by interleukin-1β in rat astrocytes

Abstract Interleukin-1β (IL-1β) is a pro-inflammatory cytokine produced in the brain by endogenous microglial cells responding to injury. Levels of IL-1β are elevated in several neurodegenerative disorders, including Alzheimers disease. IL-1β, which can act as a mitogen for astrocytes, also elicits the expression and secretion of multiple factors and paracrine ‘second messengers’ such as other cytokines, nerve growth factor, prostaglandins and nitric oxide that may in turn modulate neuronal and glial responses to injury. Utilizing giant, high-resolution two-dimensional gel electrophoresis, we have sought to more fully define the potential range of protein mediators that are secreted by astrocytes treated with IL-1β. In cultured rat astrocytes, we observe dramatic increases in the secretion of eight different protein species after 24 h of treatment with human recombinant IL-1β (1 U/ml). Seven of the proteins are also induced by tumor necrosis factor-α or basic fibroblast growth factor. Based on immunoprecipitation with specific antisera, we have identified three of these proteins as plasminogen activator inhibitor type-1, ceruloplasmin, and complement component C3. The identities of the other proteins, including the IL-1β-specific induction, are currently unknown. Characterization of these downstream modulators of IL-1β action complements gene-based approaches and will provide a better understanding of astrocyte responses to injury as well as markers for astrocyte activation in neurodegenerative diseases.

Glucocorticoid and phorbol ester effects in 3T3-L1 fibroblasts suggest multiple and previously undescribed mechanisms of glucocorticoid receptor-AP-1 interaction

A number of mechanisms have been proposed to account for glucocorticoid hormone and mitogen effects observed on the transcription of engineered exogenous target genes. However, the effect of the co-addition of these agents on the full range of endogenous responsive genes in a given cell type has not been addressed. We detected 19 metabolically labeled proteins or in vitro translation products whose synthesis was altered in 3T3-L1 fibroblasts in response to glucocorticoid hormone using two-dimensional electrophoresis on large-format gels. Co-addition of the mitogenic phorbol ester, tetradecanoylphorbolacetate (TPA), with glucocorticoid hormone resulted in independent (one species), synergistic (six species), and antagonistic (five species) effects on the glucocorticoid regulation of individual gene products, while seven other glucocorticoid regulated species were not affected. These and additional observations suggest direct and gene-specific effects of glucocorticoid receptor and AP-1 on the transcription of responsive genes, and for some of these genes the pattern of regulation is not accounted for by mechanisms described to date. In addition, the pattern of regulation of five species is consistent with a role in mediating the opposing physiological effects of glucocorticoid hormone and growth factors in these cells.

EMERGENCE OF RESISTANCE TO GLUCOCORTICOID HORMONES IN CANCER CELLS: DETECTION OF DIFFERENCES IN PROTEINS SYNTHESIZED BY GLUCOCORTICOID-SENSITIVE AND -RESISTANT MOUSE P1798 LYMPHOSARCOMA CELLS

Publisher Summary This chapter discusses the emergence of resistance to glucocorticoid hormones in cancer cells and detection of differences in proteins synthesized by glucocorticoid-sensitive and resistant mouse P1798 lymphosarcoma cells. The changes in the kinds of proteins made that accompany the emergence of resistance to glucocorticoids are observed by examining individual proteins separated by two-dimensional polyacrylamide gel electrophoresis. By increasing the size of the first and second dimension gel systems by 2.5-fold, the resolution is approximately doubled, allowing the detection of >2000 separate protein peaks. In comparing glucocorticoid-sensitive versus glucocorticoid-resistant cells, there are at least 20 proteins whose synthetic rates differ significantly. These differences are retained through at least three tumor passages, suggesting that the changes are relatively stable. Some proteins are detectable only in the sensitive line whereas others are detectable only in the resistant line. Thus, the possibility remains open that resistance may be conferred through either an increase or a decrease in the amounts of one or more proteins. The chapter discusses the localization of the protein differences to specific subcellular compartments for determining which proteins are membrane-associated.