Paul R. Reynolds

BMP-6 Is an Autocrine Stimulator of Chondrocyte Differentiation

While parathyroid hormone‐related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13‐day embryos, and the role of bone morphogenetic protein‐6 (BMP‐6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP‐6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10−7 M) at the time of plating completely abolished BMP‐6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP‐6 and type X collagen mRNA, with BMP‐6 again preceding type X collagen expression. The addition of exogenous BMP‐6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP‐6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP‐6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP‐6, it indirectly regulates Ihh expression through BMP‐6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP‐6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP‐6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP‐6.

Increased Levels of Tumor Necrosis Factor-α and Interleukin-6 Protein and Messenger RNA in Human Peripheral Blood Monocytes due to Titanium Particles*

Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from volunteers and blood that had been donated to the American Red Cross and were cultured in the presence of titanium particles (one to three micrometers in diameter). There were consistent dose-dependent increases in the production of TNF-&agr; (tumor necrosis factor-&agr;) and IL-6 (interleukin-6) protein, with the greatest stimulation generally observed with a concentration of 6 x 105 to 6 x 106 particles of titanium per milliliter. The level of TNF-&agr; was the greatest (fifty to 1000 times greater than the control level) after eight hours of exposure to titanium particles; the level of IL-6 was two to five times greater than the control level after sixteen hours of exposure. These increases were similar to those observed after stimulation with lipopolysaccharide and depended on de novo synthesis rather than on release from intracellular stores. The production of TNF-&agr; was inhibited in a dose-dependent manner by the translational inhibitor cycloheximide and the transcriptional inhibitor actinomycin D, indicating the requirement for both mRNA (messenger RNA) and protein synthesis for the induction of cytokine synthesis by titanium particles. Although the increase in the levels of cytokine mRNA in response to titanium was rapid (thirty to ninety minutes), the increase in the level of TNF-&agr; mRNA preceded that of IL-6 mRNA. The level of TNF-&agr; mRNA was the greatest at ninety minutes and the level of IL-6 mRNA was the greatest at three hours. After stimulation with titanium particles, the level of TNF-&agr; mRNA was increased as much as fivefold and the level of IL-6 mRNA, as much as twelvefold. CLINICAL RELEVANCE: Awareness of the importance of wear debris particles in cytokine-induced bone resorption has resulted in improvements in the designs of implants and in operative techniques to reduce wear of components. The present study further elucidates the biological mechanisms involved in periprosthetic osteolysis. Titanium-stimulated biosynthesis of the cytokines TNF-&agr; and IL-6, which are both potent stimulators of bone resorption, requires increases in the synthesis of both mRNA and protein by monocytes. An understanding of the complex mechanisms of the induction of cytokine synthesis by particles of wear debris will facilitate the design of pharmacological agents to control periprosthetic bone resorption. These agents, in combination with other efforts to reduce the generation of wear debris, may improve the longevity of orthopaedic implants.

PTHrP Modulates Chondrocyte Differentiation through AP-1 and CREB Signaling

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage.

Procaryotic and eucaryotic cells possess mechanisms for arresting cell division in response to DNA damage. Eucaryotic cells arrest division in the G2 stage of the cell cycle, and various observations suggest that this arrest is necessary to ensure the completion of repair of damaged DNA before the entry of cells into mitosis. Here, we provide evidence that the Saccharomyces cerevisiae RAD9 gene, mutations of which confer sensitivity to DNA-damaging agents, is necessary for the cell cycle arrest phenomenon. Our studies with the rad9 delta mutation show that RAD9 plays a role in the cell cycle arrest of methyl methanesulfonate-treated cells and is absolutely required for the cell cycle arrest in the temperature-sensitive cdc9 mutant, which is defective in DNA ligase. At the restrictive temperature, cell cycle progression of cdc9 cells is blocked sometime after the DNA chain elongation step, whereas cdc9 rad9 delta cells do not arrest at this point and undergo one or two additional divisions. Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis. The rad9 delta mutation does not affect the frequency of spontaneous or UV-induced mutation and recombination, suggesting that RAD9 is not directly involved in mutagenic or recombinational repair processes. The RAD9 gene encodes a transcript of approximately 4.2 kilobases and a protein of 1,309 amino acids of Mr 148,412. We suggest that RAD9 may be involved in regulating the expression of genes required for the transition from G2 to mitosis.

BMP signaling stimulates chondrocyte maturation and the expression of Indian hedgehog

Mutant BMP receptors were transfected into cultured embryonic upper sternal chrondrocytes using retroviral vectors to determine if BMP signaling is required for chondrocyte maturation and the expression of a key regulatory molecule, Indian hedgehog (Ihh). Chondrocytes infected with replication competent avian retroviruses (RCAS) viruses carrying constitutive active (CA) BMPR‐IA and BMPR‐IB had enhanced expression of type X collagen and Ihh mRNA. Addition of PTHrP, a known inhibitor of chondrocyte maturation, abolished the expression of type X collagen, BMP‐6, and Ihh mRNAs in control cells. In contrast, PTHrP treated cultures infected with of CA BMPR‐IA or CA BMPR‐IB had low levels of BMP‐6 and type X collagen, but high levels of Ihh expression. Although dominant negative (DN) BMPR‐IA had no effect, DN BMPR‐IB inhibited the expression of type X collagen and BMP‐6, and decreased alkaline phosphatase activity, even in the presence of exogenously added BMP‐2 and BMP‐6. DN BMPR‐IB also completely blocked Ihh expression. Overall, the effect of DN BMPR‐IB mimicked the effects of PTHrP. To determine if there is an autocrine role for the BMPs in chondrocyte maturation, the cultures were treated with noggin and follistatin, molecules that bind BMP‐2/‐4 and BMP‐6/‐7, respectively. While noggin and follistatin inhibited the effects of recombinant BMP‐2 and BMP‐6, respectively, they had only minimal effects on the spontaneous maturation of chondrocytes in culture, suggesting that more than one subgroup of BMPs regulates chondrocyte maturation. The results demonstrate that: (i) BMP signaling stimulates chondrocyte maturation; (ii) BMP signaling increases Ihh expression independent of maturational effects; and (iii) BMP signaling can partially overcome the inhibitory effects of PTHrP on maturation.

Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice

RATIONALE The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity. OBJECTIVES To test the inducibility of lung defenses against bacterial challenge. METHODS Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol. MEASUREMENTS AND MAIN RESULTS Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48-72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid. CONCLUSIONS We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.

A Role for the BMP Antagonist Chordin in Endochondral Ossification

Bone morphogenetic proteins (BMPs) are ubiquitous regulators of cellular growth and differentiation. A variety of processes modulate BMP activity, including negative regulation by several distinct binding proteins. One such BMP antagonist chordin has a role in axis determination and neural induction in the early embryo. In this study, a role for chordin during endochondral ossification has been investigated. During limb development, Chordin expression was detected only at the distal ends of the skeletal elements. In cultured embryonic sternal chondrocytes, Chordin expression was related inversely to the stages of maturation. Further, treating cultured chondrocytes with chordin interfered with maturation induced by treatment with BMP‐2. These results suggest that chordin may negatively regulate chondrocyte maturation and limb growth in vivo. To address this hypothesis, chordin protein was expressed ectopically in Hamburger‐Hamilton (HH) stage 25‐27 embryonic chick limbs. The phenotypic changes and alteration of gene expression in treated limbs revealed that overexpression of chordin protein delayed chondrocyte maturation in developing skeletal elements. In summary, these findings strongly support a role for chordin as a negative regulator of endochondral ossification.

Modulation of the Production of Cytokines in Titanium-Stimulated Human Peripheral Blood Monocytes by Pharmacological Agents. The Role of cAMP-Mediated Signaling Mechanisms*†

Cytokines secreted by activated macrophages play a role in the development of osteolysis adjacent to prosthetic joints. To determine whether the synthesis of cytokines can be inhibited by pharmacological agents, we studied the role of the cAMP-protein kinase A signal transduction pathway in the synthesis of interleukin-6 and tumor necrosis factor-&agr; and examined the effect of potential pharmacological regulators of this pathway in human peripheral blood monocytes stimulated with titanium particles. Dibutyryl cAMP enhanced the synthesis of interleukin-6 by titanium-stimulated monocytes and resulted in a marked increase (maximum, seventyfold) in the synthesis of interleukin-6 even in the absence of titanium particles. However, the active analogs (agonists) of cAMP, dibutyryl cAMP and Sp cAMP, inhibited the production of tumor necrosis factor-&agr; by titanium-stimulated monocytes (the maximum effects resulted in complete inhibition), while the cAMP antagonist, Rp cAMP, enhanced the production of tumor necrosis factor-&agr;. Additional agents that alter the intracellular levels of cAMP were examined for their effects on the synthesis of cytokines. Prostaglandins E1 and E2 were potent inhibitors of the synthesis of tumor necrosis factor-&agr; but stimulated the synthesis of interleukin-6. In contrast, indomethacin enhanced the stimulatory effects of titanium particles on tumor necrosis factor-&;, resulting in a more than threefold increase in the maximum levels of tumor necrosis factor-&agr;. Phosphodiesterase inhibitors, such as isobutyryl methylxanthine and pentoxifylline, which increase intracellular levels of cAMP, caused a decrease in the production of tumor necrosis factor-&agr; and an increase in the production of interleukin-6. In contrast, the fluoroquinolone antibiotic ciprofloxacin, which is also a phosphodiesterase inhibitor, caused a dose-dependent inhibition of the synthesis of both tumor necrosis factor-&agr; and interleukin-6 by titanium-stimulated monocytes, suggesting that ciprofloxacin suppresses the synthesis of interleukin-6 through a mechanism that is independent of cAMP. CLINICAL RELEVANCE: Pharmacological agents, in combination with efforts at reducing the generation of wear debris, may lead to novel therapeutic strategies to prevent the loosening of implants. Alteration of the synthesis of cytokines in response to pharmacological manipulation of the cAMP-protein kinase A signaling pathway suggests that these events are regulated by normal physiological mechanisms in titanium-stimulated human peripheral blood monocytes rather than by an irreversible toxic response. The differential response of tumor necrosis factor-&agr; and interleukin-6 to agents that alter intracellular levels of cAMP indicates that these two cytokines are independently regulated. Thus, pharmacological strategies to inhibit the release of cytokines may need to involve multiple agents to inhibit diverse regulatory pathways. Basic-science studies identifying the pathways involved in titanium-mediated synthesis of cytokines are essential for the efficient design and selection of inhibitory agents.

The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin conjugating enzyme and is required for DNA repair, DNA‐damage‐induced mutagenesis and sporulation. Here, we show that RAD6 and the rhp6+ gene from the distantly related yeast Schizosaccharomyces pombe share a high degree of structural and functional homology. The predominantly acidic carboxyl‐terminal 21 amino acids present in the RAD6 protein are absent in the rhp6(+)‐encoded protein; otherwise, the two proteins are very similar, with 77% identical residues. Like rad6, null mutations of the rhp6+ gene confer a defect in DNA repair, UV mutagenesis and sporulation, and the RAD6 and rhp6+ genes can functionally substitute for one another. These observations suggest that functional interactions between RAD6 (rhp6+) protein and other components of the DNA repair complex have been conserved among eukaryotes.

Up-regulation of AMP-activated Kinase by Dysfunctional Cystic Fibrosis Transmembrane Conductance Regulator in Cystic Fibrosis Airway Epithelial Cells Mitigates Excessive Inflammation

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, ΔF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-α and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and ΔF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.