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R L Jilka

Publications and source records attributed to R L Jilka.

At least 55 records · Page 3Linked to original sources
PubMed1989

Stimulation of collagenolytic enzyme release from cultured bone cells of normal and osteopetrotic (mi/mi) mice by parathyroid hormone and lipopolysaccharide.

Collagenolytic enzyme release from bone cells was studied using cultured calvarial cells which are capable of degrading calcified and noncalcified collagen (cells from normal mice) or only noncalcified collagen (cells from osteopetrotic (mi/mi) mice). Treatment of cells from either normal or mi/mi mice with parathyroid hormone (PTH) or lipopolysaccharide (LPS) resulted in the appearance of latent collagenolytic enzyme activity in the medium. Chromatography of media from cells from normal mice treated with PTH on lysine-Sepharose resulted in the separation of latent collagenase and latent gelatinase. Further characterization of the enzymes showed that they were similar to those previously isolated from media of calvaria cultured with heparin. Collagenase activity of media of cells from normal or mi/mi mice treated with PTH or LPS yielded identical elution patterns upon chromatography on lysine-Sepharose. These results show that the mi allele has no effect on regulation of latent collagenolytic enzyme release. The results show that the mi allele has no effect on regulation of latent collagenolytic enzyme release. The data also suggest that previously described differences between PTH- and LPS-stimulated collagen degradation in cultured calvaria are due to factors other than differences in the ability of these agents to stimulate the release of collagenolytic enzymes.

Animals↗
PubMed1989

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone.

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20-30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.

Animals↗
PubMed1989

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

Animals↗
PubMed1989

Functional and molecular changes in colony stimulating factor secretion by osteoblasts.

Two populations of primary osteoblasts and the cloned murine osteoblast cell line MC3T3 were activated with osteotropic agents and the conditioned media tested for the presence of macrophage colony stimulating factor (M-CSF). Differences in ability of the three populations to secrete the cytokine as well as in the efficacy of the activating agents used to induce it were observed. The ability of these agents to modulate M-CSF mRNA levels was also examined. The data indicate that osteoblasts do not respond uniformly and the reasons for this are discussed. Osteoblasts may exert major regulatory influences on hematopoiesis via CSF secretion as well as function in their more traditional role as the cells responsible for new bone formation.

Animals↗
PubMed1987

Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57Bl/6J mice.

The osteopetrotic, microphthalmic (mi/mi) mouse lacks functional osteoclasts and has also been reported to be deficient in mast cells and natural-killer (NK) cells. The later deficiencies could be secondary to the osteopetrotic marrow, or a direct result of the mi allele. Therefore, heterozygotes were examined for these cell types, since these mice do not exhibit osteopetrosis. Adult +/mi animals have approximately 50%, and mi/mi animals examined by histologic techniques or tissue histamine levels have 0-10%, of the peritoneal, dermal, and intestinal mast cells compared with that of +/+ animals. Leukocyte histamine, indicative of the number of basophils, demonstrates the same pattern. Histamine content per mast cell in +/+ and +/mi animals is identical. The number of large granular lymphocytes (LGL) in splenic leukocyte preparations from +/mi animals is 50% that of +/+ animals, and these cells are undetectable in preparations from mi/mi mice. NK activity against YAC-1 cells paralleled the number of LGL present. The resorptive response of neonatal calvaria to parathyroid hormone was delayed in the case of cultured +/mi bone compared with that of +/+ bone, but the final rate of calcium release was identical. These data indicate that 1) the presence of one mi allele can affect the development of four distinct cell types, and 2) osteopetrosis alone does not account for the lack of mast cells, basophils, and NK cells in mi/mi mice.

Alleles↗
PubMed1986

Formation and secretion of fragments of parathormone. Identification of cleavage sites.

Monolayer cultures of bovine parathyroid cells or fresh gland slices were incubated with radioactive amino acids in order to study the formation and metabolism of parathormone (PTH). PTH, secretory protein I, and COOH-terminal fragments of PTH were all released into media within 30 min, most strongly in the first hour after synthesis. Peptides in tissue, cells, and media were separated using reverse-phase high performance liquid chromatography. In eluates of media, six radioactive peaks were prominent. The first four and the sixth were immunoreactive in a COOH-terminal specific PTH radioimmunoassay, but only the sixth was reactive in an NH2-terminal specific assay. Under conditions where recovery of PTH(1-34) was quantitative, gel filtration of media was used to show that no NH2-terminal fragments of PTH were secreted. Sequence analyses of secreted COOH-terminal peptides indicated that the NH2 termini of the first three peaks corresponded to residues 43, 37, and 34 of PTH. The fourth peak contained a mixture of two peptides with NH2 termini at residues 24 and 28 of PTH. The fifth could not be identified; the sixth was PTH. Cleavages at the 23-24 bond of PTH occurred within minutes of the formation of PTH itself, and the other peptides were formed more slowly. Mandatory cleavage of PTH at the 23-24 peptide bond would destroy the biological activity of the hormone on kidney and bone, a situation consistent with the possibility that intracellular PTH metabolism participates in secretory regulation. The results showed that different peptides were generated in parathyroid cells than were previously shown to be produced by cathepsin B or D. The results suggest that the proteolytic pathway which results in the secretion of PTH fragments is nonlysosomal in nature.

Amino Acid Sequence↗
PubMed1986

Formation of parathormone 8-34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist.

It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.

Amino Acid Sequence↗
PubMed1986

Parathyroid hormone-stimulated development of osteoclasts in cultures of cells from neonatal murine calvaria.

Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 mu in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRAPase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRAPase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 mu in diameter, sometimes multinucleated, and some were spread 100 to 200 mu in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH-stimulated development of TRAPase-positive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 mu diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH.

Acid Phosphatase↗
PubMed1986

Are osteoblastic cells required for the control of osteoclast activity by parathyroid hormone?

Osteoclasts cultured from the endosteal surface of marrow appear to require osteoblasts for PTH to stimulate their resorptive activity, but the contribution of preosteoclasts to these observations has not been ruled out. Other data indicate that an osteoblast-derived factor can influence osteoclast activity, or confer sensitivity of osteoclasts to PTH. Acid phosphatase content of osteoclasts formed in osteoblast-free cultures of marrow cells is stimulated by PTH, but resorption is not. Also, there are conflicting data on the presence of a PTH receptor on osteoclasts. Thus, while PTH may act directly on preosteoclasts, or even on osteoclasts, to enhance resorptive capability, it seems evident that an osteoblastic factor mediates the PTH stimulation of osteoclastic resorptive activity.

Animals↗
PubMed1985

Evidence for two pathways for stimulation of collagenolysis in bone.

The effect of parathormone (PTH), lipopolysaccharide (LPS), or interleukin-1 (IL-1) on calcium release and collagen degradation in bone was examined in vitro using labeled neonatal calvaria of normal mice and also of osteopetrotic microphthalmic (mi/mi) mice that have defective osteoclasts. All three agents stimulated calcium release from normal bone but not from mi/mi bone. PTH stimulated the degradation of both noncalcified and calcified collagen in normal bone as well as the degradation of noncalcified collagen in mi/mi bone. However, LPS and IL-1 only stimulated the degradation of calcified collagen in normal bone. One-half maximal stimulation of noncalcified collagen degradation in normal or mi/mi bone was achieved by about 3 nM PTH compared with about 1 nM PTH for that of calcium release from normal bone. While calcitonin (CT) and leupeptin inhibited calcium release and thereby the degradation of calcified collagen, neither agent inhibited PTH-stimulated noncalcified collagen degradation in normal or mi/mi bone. The data indicate the existence of two pathways that lead to collagen degradation in bone. One is intimately connected with the resorptive process stimulated by a variety of agents, and is probably mediated by osteoclasts. A second mechanism is sensitive only to PTH and appears to be associated with nonosteoclastic cells since it can operate under conditions in which osteoclasts are thought to be inactive or are inhibited.

Acetazolamide↗
PubMed1985

Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice.

The content of carbonic anhydrase isozymes I (CA I) and II (CA II) in red blood cells and bone of osteopetrotic microphthalmic (mi/mi) mice was analyzed. Monospecific rabbit polyclonal antibodies against purified rat carbonic anhydrase I or II detected both isozymes in hemolysates of both normal and mi/mi mice. Total carbonic anhydrase activity measurements of hemolysates from normal or mi/mi mice were identical. A procedure based on bromopyruvate inactivation was devised to measure the relative contributions of CA I and II isozymes to the carbon dioxide hydration activity of hemolyzates. CA II dominates the observed activity of hemolysates of normal and mi/mi mice. Immunohistochemical studies showed that CA II was present in osteoclasts of tibial and calvarial bones of both normal and mi/mi mice. Thus, in contrast to the several cases of inherited osteopetrosis in humans, there is no lack of active CA II in erythrocytes of mi/mi mice. The absence of CA II, therefore, does not appear to play a role in the etiology of osteopetrosis in the mi/mi mouse.

Animals↗
PubMed1984

Inhibition of parathormone-stimulated bone resorption by type I interferon.

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.

Animals↗
PubMed1983

A collagenolytic response to parathormone, 1,25-dihydroxycholecalciferol D3, and prostaglandin E2 in bone of osteopetrotic (mi/mi) mice.

Stimulators of bone resorption, such as PTH, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], or prostaglandin E2 (PGE2), do not cause calcium release from cultured calvaria of the genetically determined osteopetrotic microphthalmic (mi/ mi) mouse, due to a defect in the function of osteoclasts. To investigate the capacity of cells of mi/mi bone to degrade collagen, calvaria of 1- to 3-day-old normal and mi/mi littermates were labeled in vivo with [3H]proline 16 h before removal, followed by culture in resorption medium. PTH, 1,25-(OH)2D3, and PGE2 stimulated the release of 3H-labeled material into the culture medium from both normal and mi/mi calvaria. The labeled substance released was of collagenous origin, as indicated by its content of hydroxyproline and susceptibility to collagenase. PTH also stimulated the release of 3H-labeled materials from normal calvaria labeled in vivo 112 h before the mice were killed, but had little or no effect on 3H release from the mi/mi bone, indicating that only noncalcified collagen is susceptible to hormone-stimulated degradation in osteopetrotic bone. We conclude that a portion of the hormone-stimulated resorptive mechanism, namely collagenolysis, is functional in bone of mi/mi mice. This result helps to pinpoint the resorptive defect in mi/ mi bone to a failure to dissolve mineral, rather than a more general phenomenon of failure to remove both mineral and matrix.

Animals↗
PubMed1983

Cleavage of parathyroid hormone to the 1-34 and 35-84 fragments by cathepsin D-like activity in bovine parathyroid gland extracts.

We have obtained a crude enzyme preparation from bovine parathyroid gland homogenates which when incubated with PTH, cleaves the hormone into two major fragments. Isolation and chemical analysis has led to the identification of these peptides, the 1-34 fragment and the 35-84 fragment. Digestion of PTH was totally inhibited by the inclusion of the cathepsin D inhibitor, pepstatin, in the enzyme digest. A comparison of the digest obtained using the crude enzyme fraction vs. digestion of PTH by purified bovine cathepsin D led to the findings that the same peptide products were formed in each case. The natural 1-34 hormone fragment derived from the procedure has been determined to be fully biologically active in a bone resorption system.

Amino Acids↗
PubMed1982

Formation of bone by isolated, cultured osteoblasts in millipore diffusion chambers.

Osteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6-7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an "inducer." After 20 days, 11 of 18 chambers containing to osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.

Animals↗
PubMed1981

Role of phosphodiesterase in the parathormone-stimulated adenosine 3',5'-monophosphate response in bone cell populations enriched in osteoclasts and osteoblasts.

The stimulation of cAMP formation by parathormone was assessed in isolated bone cell populations enriched in either osteoclast-like (CL) or osteoblast-like (BL) cells. In the presence of the potent phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), at 37 C, the CL cells produced at least 20% the amount of cAMP formed by the BL cells, but only about 5% when the inhibitor was omitted. The rate of cAMP production in response to parathormone was measured at 23 C in order to obtain a better indication of the capacity of the cells to generate this nucleotide. Under these conditions, the CL cells produced cAMP at a rate of 50-60% that of the BL cells regardless of whether IBMX was present. The phosphodiesterase activity in CL cell extracts was twice that in BL cell extracts, and IBMX inhibited this activity. These results show that when an effective inhibitor of phosphodiesterase is present, the CL cells exhibit a significant response to parathormone in terms of cAMP production. The possibility that the cAMP response in the CL cells was due to contaminating BL cells (which would have to be about 50% of the total cell number) was ruled out by the demonstration that citrate decarboxylating activity, a marker for the BL cells, was low and was not inhibited by parathormone. These data reinforce the earlier contention that parathormone directly stimulates adenylate cyclase in both osteoclasts and osteoblasts and activates bone resorption via cAMP as second messenger.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Homologous and heterologous control of bone cell adenosine 3',5'-monophosphate response to hormones by parathoromone, prostaglandin E2, calcitonin, and 1,25-dihydroxycholecalciferol.

Desensitization of hormone-stimulated cAMP response to parathormone, prostaglandin E2, and calcitonin was characterized in isolated bone cells. Osteoclast- and osteoblast-like cells derived from mouse calvaria were incubated for up to 24 h with agents that cause bone resorption (parathormone, prostaglandin E2, or 1,25-dihydroxycholecalciferol) or with calcitonin, which inhibits bone resorption. The cells were then exposed to parathormone, prostaglandin E2, or calcitonin, and cAMP formation was quantified. Homologous desensitization (refractoriness to the second exposure to the same hormone) occurred for each hormone and was essentially complete after 30 min of preincubation. Heterologous desensitization to parathormone could be produced in both cell types by 1,25-dihydroxycholecalciferol and prostaglandin E2, but not by calcitonin. Heterologous desensitization was of lesser magnitude, requiring more than 8 h to reach significant proportions. In contrast, the cAMP response to calcitonin and prostaglandin E2 could not be altered by preincubation with any other hormone. Dibutyryl cAMP or 3-isobutyl-1-methylxanthine, which are bone-resorptive agents, increased cellular cAMP and caused a slowly developing refractoriness to parathormone, prostaglandin E2, and calcitonin. Calcium (5 mM), an agent which mimics the biochemical action of parathormone in the bone cells but does not affect cAMP similarly desensitized the cells to parathormone, but not to calcitonin and only slightly to prostaglandin E2. These data suggest that heterologous desensitization of bone cells to parathormone results from the initiation of resorption by any agent regardless of whether cAMP is formed during activation of the cells. According to this concept, homologous desensitization to parathormone could involve two components: a rapid phase (< 30 min) and a long term phase, possibly stemming from initiation of resorption.

Animals↗