Osteoclast formation and bone resorption are inhibited by megakaryocytes.

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.

Circulating levels of interleukin-6 and its soluble receptor in patients with head injury and fracture.

There is evidence that fractures heal more rapidly in patients with head injury. We measured the circulating level of interleukin-6 (IL-6) and its soluble receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) in serum from patients who had sustained a head injury with and without fracture and compared these with levels found in control subjects. Within 12 hours of injury the serum level of IL-6 was significantly higher in patients with head injury and fracture compared with the control group. Levels of IL-6 were also significantly higher in patients with head injury and fracture compared with fracture only. While there was no significant difference in circulating levels of sIL-6R in the initial samples they were increased one week after surgery in patients with head injury and fracture and with head injury only. In addition, reduced levels of sgp130 in patients with head injury with and without fracture indicated a possible reduction of the inhibitory effect of this protein on the activity of IL-6. Our study suggests that IL-6 may be involved in altered healing of a fracture after head injury.

Circulating levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in patients with severe head injury.

There is evidence to suggest that fractures heal more rapidly in patients with a head injury as a result of systemic factors released from the site of this injury. We have measured the circulating level of insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3) in serum because of their known involvement in the stimulation of the activity of osteoblasts and the healing of fractures. The serum level of IGF-1 was significantly lower in patients with both head injury and fracture and fracture only compared with that in healthy volunteers (p < 0.01 and p < 0.02, respectively). The level of IGFBP-3 was also significantly lower in patients with both head injury and fracture (p < 0.01). Our findings showed, however, that the level of IGF-1 and IGFBP-3 varied from week to week in both the patients and healthy control subjects. These results indicate that the levels of circulating IGF-1 and IGFBP-3 are unlikely to be responsible for the altered healing of fractures seen in conjunction with head injury.

Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.

Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.

AIMS/HYPOTHESIS: Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. METHODS: Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. RESULTS: The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. CONCLUSION/INTERPRETATION: Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.

The SH3 and BH domains of the p85alpha adapter subunit play a critical role in regulating class Ia phosphoinositide 3-kinase function.

We have investigated the role of the SH3 and BH domains in the function of the p85alpha adapter/regulatory subunit of PI 3-kinase. In these studies epitope-tagged adapter subunit constructs containing wild-type p85alpha, p85alpha lacking the SH3 domain (deltaSH3-p85alpha), or p85alpha lacking the Rac-GAP/BCR homology (BH) domain (deltaBH-p85alpha) were coexpressed with either the p110alpha or p110beta PI 3-kinase catalytic subunit in HEK293 cells. The deletion of either BH or SH3 domains had no effect on the intrinsic activity of the PI 3-kinase heterodimers. However, the ability of activated Rac to stimulate PI 3-kinase activity was only observed in heterodimers containing the p85alpha and deltaSH3-p85alpha, indicating that rac binding to the BH domain is responsible for rac-induced stimulation of class Ia PI 3-kinase. We also investigated the effect of SH3 and BH domain deletion on the ability of insulin to induce recruitment of these constructs into phosphotyrosine-containing signaling complexes. We find that p85alpha expressed alone is poorly recruited into such signaling complexes. However, when coexpressed with catalytic subunit, the p85alpha adapter subunit is recruited to an extent similar to that of endogenous p85alpha. Maximal insulin stimulation caused a similar level of recruitment of p85alpha, deltaSH3-p85alpha, and deltaBH-p85alpha to signaling complexes when these adapter subunits were coexpressed with catalytic subunit. However, there was a higher level of basal association of the deltaSH3-p85alpha and deltaBH-p85alpha with tyrosine-phosphorylated proteins, meaning that the insulin-induced fold increase in recruitment was lower for these forms of the adapter. These results indicate that the N-terminal domains of p85alpha play a critical role in the way the adapter subunit responds to growth factor stimulation.

Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone.

Degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. The balance between MMPs and TIMPs may therefore be a determinant of normal bone turnover, and imbalance could thus lead to reduced organization of bone structure. To test this hypothesis, the cellular expression of MMPs and TIMP-1 was investigated by immunohistochemistry in human neonatal rib and osteophytic and heterotopic bone; these differ in their structure, with heterotopic bone showing the least and normal rib the most organized development. In all samples, high levels of MMPs were expressed. Collagenase and stromelysin-2 were detected in chondrocytes, osteoblasts, and osteoclasts, whereas gelatinase-B was confined to osteoclasts and mononuclear cells. Matrix-associated stromelysin-1 was present in fibrous tissue and osteoid. In contrast, the expression of TIMP-1 varied markedly between the three types of bone. In heterotopic bone only occasional low level TIMP-1 expression was detected in chondrocytes and osteoblasts. Osteophytic bone showed varying levels of TIMP-1, which was matrix-bound in fibrous tissue and cell-associated in osteoblasts, chondrocytes, and occasional mononuclear cells. In both types of bone, expression of TIMP-1 by osteoclasts was absent despite large numbers of these cells. Neonatal rib bone showed consistent expression of TIMP-1, particularly in chondrocytes, osteoblasts, and lining cells. In contrast to pathological bone, many osteoclasts were TIMP-1 positive. These results suggest that, in heterotopic and osteophytic bone, the low levels of TIMP-1, and in particular its absence in osteoclasts, may partly explain the more poorly organized bone formation in these pathological bone samples. Furthermore, TIMP-1 may play a role in the regulation of bone modeling and remodeling in normal developing human bone.

Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells.

Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.