Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis.

Regulation of CRIg expression and phagocytosis in human macrophages by arachidonate, dexamethasone, and cytokines.

Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg(+) macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-γ, IL-4, and transforming growth factor-ß1 decreased CRIg expression on macrophages. This down- and up-regulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation.

A role for the phosphatidylinositol 3-kinase--protein kinase C zeta--Sp1 pathway in the 1,25-dihydroxyvitamin D3 induction of the 25-hydroxyvitamin D3 24-hydroxylase gene in human kidney cells.

The molecular mechanisms that underlie non-genomic induction of the 25-hydroxyvitamin D3 24-hydroxylase (CYP24) gene promoter by the steroid hormone, 1,25-Dihydroxyvitamin D3 (1,25D), are poorly understood. Although we have previously identified a functional inverted GC-box in the early promoter at -113/-105 bp, it is not known whether this site is important for 1,25D induction of the promoter. Using transfected human embryonic kidney (HEK) 293T cells, we now report the functional characterisation of the GC-box and that 1,25D induction of the promoter requires PI3-kinase, PKCzeta and Sp1 but not Sp3. The data show that 1,25D rapidly stimulates PI3-kinase activity which is required for the activation of PKCzeta and the phosphorylation of Sp1. The effects of the PI3-kinase inhibitor, LY294002, and a dominant negative PKCzeta mutant on 1,25D induction of wild-type and a GC-box mutated CYP24 promoter constructs are consistent with the Sp1 site being the target of both kinases. However, these kinases are not required for basal expression of the CYP24 promoter. The data establish a novel non-genomic mechanism which couples 1,25D to the induction of CYP24 gene transcription via the PI3-kinase--PKCzeta--Sp1 pathway acting through the GC-box.

A novel beta-oxa polyunsaturated fatty acid downregulates the activation of the IkappaB kinase/nuclear factor kappaB pathway, inhibits expression of endothelial cell adhesion molecules, and depresses inflammation.

Several novel polyunsaturated fatty acids (PUFAs) that contain either an oxygen or sulfur atom in the beta-position were found to exhibit more selective antiinflammatory properties than their natural PUFA counterparts. One of these, beta-oxa-23:4n-6, unlike natural PUFAs, lacked ability to stimulate oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC) and E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. In addition, beta-oxa-23:4n-6 inhibited acute and chronic inflammatory responses in mice as well as the upregulation of adhesion molecule expression in arterial endothelium. This action of beta-oxa-23:4n-6 required a functional 12- but not 5-lipoxygenase or cyclooxygenases, consistent with its metabolism via the 12-lipoxygenase pathway. Whereas beta-oxa-23:4n-6 did not affect the activation of mitogen-activated protein kinases by tumor necrosis factor, activation of the IkappaB kinase/nuclear factor kappaB pathway was selectively inhibited. These novel PUFAs could form the basis for a potential new class of pharmaceuticals for treating inflammatory diseases, including atherosclerosis.

Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site.

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D3 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D3) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys71-->Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at -171/-163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5'-TGTCGGTCA-3', is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.

The immunomodulatory effects of novel beta-oxa, beta-thia, and gamma-thia polyunsaturated fatty acids on human T lymphocyte proliferation, cytokine production, and activation of protein kinase C and MAPKs.

We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (beta-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of beta-oxa, beta-thia, and gamma-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated beta-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-beta production. However, beta-oxa 25:6n-3 and beta-oxa 21:4n-3 displayed lower inhibitory action on IFN-gamma production. Surprisingly, beta-oxa 23:4n-6 and beta-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The beta-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two gamma-thia PUFA tested, gamma-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but gamma-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, beta-thia 23:4n-6 and beta-thia 21:3n-6, were studied in more detail and shown to have an IC(50) of 1-2 muM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A2.

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.

Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells.

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKC beta I, epsilon, theta and zeta. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4 h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.

Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5.

The current study investigated the action of 1,25-dihydroxyvitamin D(3) (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogen-activated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction of CYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor alpha (RXR alpha) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR alpha and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR alpha and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXR alpha (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXR alpha phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.