Developmental expression of creatine kinase isoenzymes in chicken growth cartilage.

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.

Endogenously opsonized particles divert prostanoid action from lethal to protective in models of experimental endotoxemia.

We report that, in rats, the lethal consequences of high-dose endotoxin challenge are exacerbated by the intravascular administration of prostaglandin E1 but attenuated by the intravascular administration of endocytosable particles. This protection is mediated by opsonins. Nonopsonizable particles were unable to provide protection unless first pseudoopsonized with antibody directed against the CR3 (CD11b/CD18) phagocyte receptor. We show that endogenously opsonized particles can act in concert with prostaglandin E1 (putatively by elevation of neutrophil intracellular cAMP and the resultant downregulation of CR3) to completely rescue animals from the lethal late-stage sequelae of experimental endotoxemia. These data illustrate that the interaction of particles with cellular receptors can transform the overall systemic response to prostaglandin E1 from pro- to antiinflammatory. This suggests a role for multiple receptor engagement events in defining the systemic prostaglandin response and offers a rationale for developing new therapeutic modalities in the treatment of sepsis and other inflammatory diseases.

Polyionic regulation of cartilage development: promotion of chondrogenesis in vitro by polylysine is associated with altered glycosaminoglycan biosynthesis and distribution.

The development of cartilage nodules in cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24) is significantly promoted when the culture medium is supplemented with (poly-L-lysine (PL) (M(r) greater than or equal to 14K) (San Antonio and Tuan, 1986. Dev. Biol. 115: 313). Here we present findings consistent with the hypothesis that PL may promote chondrogenesis by interacting electrostatically with sulfated glycosaminoglycans (GAGs): (1) poly-L-ornithine, poly-L-histidine, poly-D,L-lysine, and lysine-containing heteropolypeptides stimulate chondrogenesis in proportion to their contents of cationic residues; (2) the effects of PL are diminished when limb mesenchyme cultures are supplemented with exogenous GAGs, including heparin, dermatan sulfate, and chondroitin sulfate; (3) in high density cultures of limb bud mesenchyme, the release of sulfated macromolecules, but not of proteins in general, into the culture medium was significantly inhibited by PL (398K M(r)) treatment, and a net increase in total GAG content of the PL-treated cultures was observed; and (4) in monolayer cultures of cells derived from other chick embryonic tissues, including liver, skeletal muscle, and calvaria, PL treatment promoted the cell layer-associated retention of sulfated GAG. These effects were not observed using the nonstimulatory, low M(r) PL (4K). Based on the above findings and those from previous studies, it is proposed that PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.