Tetracyclines induce apoptosis in osteoclasts.

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption through inhibition of matrix metalloproteinases. Here we report that some tetracyclines also induce apoptosis in rabbit osteoclasts and inhibit differentiation and activity of osteoclasts in murine osteoblast/marrow cocultures. Apoptosis of mature rabbit osteoclasts increased from 5.5 +/- 1.4% (mean +/- SD) in control cultures to 44.9 +/- 6.3% (p < 0.001) and 18.9 +/- 4.0% (p < 0.005) with CMT-3 and doxycycline (10 microg/mL), respectively. CMT-2 or CMT-5 did not alter osteoclast viability even at 25 microg/mL. In murine osteoblast/marrow cocultures over 11 days, CMT-3 and doxycycline (5 microg/mL) reduced the formation of mature osteoclasts and inhibited resorption to 21 +/- 9% (p < 0.01) and 49 +/- 4% (p < 0.01) of untreated cultures. Induction of osteoclast apoptosis is an additional property of tetracyclines that may contribute to their ability to inhibit bone resorption.

The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.

Recent identification in bone of transporters, receptors, and components of synaptic signaling suggests a role for glutamate in the skeleton. We investigated effects of glutamate and its antagonist MK801 on osteoclasts in vitro. Glutamate applied to patch clamped osteoclasts induced significant increases in whole-cell membrane currents (P<0.01) in the presence of the coagonist glycine. Agonist-elicited currents were significantly decreased after application of MK801 (100 microM, P<0.01), but MK801 had no effect on actin ring formation necessary for osteoclast polarization, attachment, and resorption. In cocultures of bone marrow cells and osteoblasts in which osteoclasts develop, MK801 inhibited osteoclast differentiation and reduced resorption of pits in dentine (3 to 100 microM; P<0.001). MK801 added early in the culture (for as little as 2-4 days) was as effective as addition for the entire culture period. Addition of MK801 for any time after day 7 of culture was ineffective in reducing osteoclast activity. Using rat and rabbit mature osteoclasts cultured on dentine or explants of mouse calvariae prelabeled with (45)Ca, we could not detect significant effects of MK801 on osteoclastic resorption. These data show clearly that glutamate receptor function is critical during osteoclastogenesis and suggest that glutamate is less important in regulating mature osteoclast activity.-Peet, N. M., Grabowski, P. S., Laketic-Ljubojevic, I., Skerry, T. M. The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.

Expression of a functional N-methyl-D-aspartate-type glutamate receptor by bone marrow megakaryocytes.

Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.

The effect of low-profile serine substitutions in the V3 loop of HIV-1 gp120 IIIB/LAI on the immunogenicity of the envelope protein.

Many microbial antigens contain powerful hypervariable epitopes that fail to induce broadly protective immunity because they dominate the immune response at the expense of more conserved but weaker epitopes. If the undesired B cell epitopes are eliminated, the immune system could be focused on the conserved epitopes and produce a stronger antibody response to conserved parts of the protein and thus become a more efficacious immunogen for a vaccine. We examined this possibility using the human immunodeficiency virus envelope glycoprotein (gp)120 IIIB/LAI and selectively replaced the amino acids from the V3 region and analyzed the overall immunogenicity of the mutant proteins after nucleic acid immunization in mice. The most variable residues of the human immunodeficiency virus type 1 gp120 V3 loop sequence were replaced with serine, which has a small uncharged hydrophilic side chain and therefore is likely to be less immunogenic than amino acids found in wildtype V3 sequences. The serine substitutions did not affect the ability of soluble CD4 to bind the mutant molecules compared with wildtype gp120 and monoclonal antibodies against both linear and discontinuous epitopes located in the V1/V2, C1, and C4 regions of the molecule. These data suggest that the V3 loop substitutions did not grossly affect the overall conformation of the envelope molecule. Immunization of CBA x BALB/c F1 mice with DNA expression plasmids for the wild-type gp120 sequence induced a predominantly IgGI antibody response with end point titers of 10(4)-5 x 10(4). The antibodies reacted only with conformationally intact gp120. Serine replacements targeted to both sides of the V3 loop had a major impact on gp120 immunogenicity, with a markedly reduced response in the majority of animals tested. Analysis of the epitope specificity of the responses suggests that N-terminal amino acids in the V3 loop contribute to the major immunodominant epitope and provides no evidence that their removal enhances immunogenicity of the conserved regions.

Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120.

We have compared the antibody response to HIV-1 gp120 type LAI in mice immunized with either a gp120 expression plasmid or with baculovirus-derived recombinant gp120 (rgp120) formulated with Freund's complete adjuvant. TiterMax, Alum, Ribi R-700, AF-A or QuilA. DNA immunization resulted in variable levels of antibody, with endpoint titres ranging from 10(4) to 10(5), whereas mice immunized with rgp 120 mixed with Ribi R-700, AF-A or QuilA produced antibody levels with endpoint titres > 10(5). Both types of immunization failed to elicit antibodies able to recognize denatured rgp120. The V3 region was immunogenic in animals immunized with nucleic acid, whereas only a few animals immunized with recombinant protein produced antibodies specific for V3 or other linear epitopes, irrespective of the adjuvant used. These data suggest that the immunogenicity of gp120 is dependent upon the mode of antigen delivery, and that in vivo expressed gp120 following nucleic acid immunization elicits, at least with respect to V3, an antibody response which more closely reflects that seen following natural infection in man.