Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Developmental regulation of expression of the α1 and α2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC α₁ and α₂ mRNAs is developmentally regulated in differentiating C2Cl2 myogenic cells. The α₁ mRNA is not detectable in the myoblast form of C2Cl2 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the α₂ mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.     

Functional expression of N-terminal truncated α-subunits of Na,K-ATPase in Xenopus laevis oocytes

N-terminal deletion mutants of Na,K-ATPase α₁ isoforms initiating translation at Met³⁴ (α₁T₁) or at Met⁴³ (α₁T₂) were expressed in X. laevis oocytes. Compared to β₃ cRNA injected controls, the co-expression of α₁wt, α₁T₁, α₁T₂ with β₃ subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K for potassium activation of the α₁T₂/β₃ Na,K-pumps is significantly higher than that of the α₁wt/β₃ or α₁T₁/β₃ Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.     

α1-Antitrypsin and α2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva

α₁-Antitrypsin, α₂-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α₁-Antitrypsin and α₂-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins.     

Lysophosphatidic acid-induced arterial wall remodeling: Requirement of PPARγ but not LPA1 or LPA2 GPCR

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 h without injury into the carotid arteries of LPA₁, LPA₂, LPA_1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and α-smooth muscle actin markers. In LPA₁, LPA₂, LPA_1&2 GPCR knockout, Mx1Cre transgenic, PPARγ^fl/−, and uninduced Mx1Cre × PPARγ^fl/− mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ^−/− knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express α-smooth muscle actin.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ were synthesized using the Wittig–Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and 1α-hydroxylated vitamin D₃ analogues.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

Recent advances in selective alpha1-adrenoreceptor antagonists as antihypertensive agents

Hypertension is one of the most serious health problems of the modern world with a continuous rise in the number of patients. Selective α₁-adrenoreceptor antagonists though have many advantages and uses in the management of arterial hypertension, their lack of specificity at the level of α₁-adr subtypes leads to multiple side effects. Existence of multiple α₁-adr subtypes holds great promise for the discovery and development of more specific and selective drug molecules, targeting only one α₁-adr subtype at a time and thus relative freedom from side effects. Herein, the research done on the discovery and evaluation of a variety of chemically diverse structures as selective antagonists of α₁-adr and α₁-adr subtypes in recent years has been reviewed.     

EFFECT OF ULCERATIVE COLITIS ACTIVITY ON PLASMA CONCENTRATION OF TRANSFORMING GROWTH FACTOR β1

Enhanced expression of transforming growth factor-β₁(TGF-β₁) demonstrated in human colonic mucosa of patients with ulcerative colitis (UC), indicates its possible significance in the pathogenesis of this disease. The aim of this study was to evaluate plasma TGF-β₁concentration in patients with different degrees of colonic mucosal injury, as a possible indicator of ulcerative colitis activity. TGF-β₁concentration was measured with an enzyme immunoassay (EIA) in plasma of 45 patients with endoscopically confirmed UC. Values observed in UC patients (40.5±15.9 ng/ml) were significantly higher than in healthy people (18.3±11.6 ng/ml) and higher than in patients with irritable colon syndrome (ICS), (20.5±13.6 ng/ml). The highest plasma TGF-β₁(58.6±112.1 ng/ml) was in patients with the severe UC course. TGF-β₁level analysed in all UC patients revealed significant positive correlation with scored degree of mucosal injury (r=0.396;P<0.01). Among other possible laboratory markers of the disease activity, only C-reactive protein concentration demonstrated significant correlation. Enhanced production of TGF-β₁can be related to inflammation activity. Measurement of plasma TGF-β₁may be considered as a biomarker of the disease activity.     

Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway

Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α₁-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α₁-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca²⁺ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α₁-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α₁-adrenoceptor (260 bp) with higher identity with α_1D and α_1B-adrenoceptors from different species. These results suggest that α₁-adrenoceptors in the venom secreting cells are probably coupled to a G_q protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.     

1α,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1α,25(OH)₂D₃ and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1α,25(OH)₂D₃ regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1α,25(OH)₂D₃-binding protein ERp60, phospholipase A₂ (PLA₂), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1α,25(OH)₂D₃ (10⁻⁸ M), which binds ERp60, activating PLA₂, and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-β1 stored in the ECM as large latent TGF-β1 complexes, consisting of latent TGF-β1 binding protein, latency associated peptide, and latent TGF-β1. Others have shown that MMP-2 specifically activates TGF-β2. TGF-β1 regulates 1α,25(OH)₂D₃-production, providing a mechanism for local control of growth factor activation. 1α,25(OH)₂D₃ activates PKCα in the PM via ERp60-signaling through PLA₂, lysophospholipid production, and PLCβ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCζ. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1α,25(OH)₂D₃, PKCζ activity is decreased and PKCα is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

Interaction of the ubiquitin carboxyl terminal esterase L1 with α2-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation

Neuroprotective effects of α₂-adrenergic receptor (AR) agonists are mediated via the α_2AAR subtype, but the molecular mechanisms underlying these actions are still not elucidated. A two-hybrid screen was performed to identify new proteins that may control α₂AR receptor function and trafficking. This screen identified the ubiquitin carboxyl-terminal hydrolase-L1 (Uch-L1), a protein associated with Parkinson's disease, as α₂AR interacting protein. This interaction was confirmed and evaluated by GST pull down assays demonstrating that Uch-L1 binds preferentially to the α_2AAR subtype and only with less affinity to α_2BAR and α_2CAR. Co-immunoprecipitation of epitope-tagged proteins confirmed the specificity of this interaction in vivo. Moreover, co-transfection of a truncated G-protein coupled receptor kinase-DNA preventing α₂AR phosphorylation led to an increased signal-strength of coimmunoprecipitated Uch-L1. Confocal laser microscopy showed that interaction of α_2AAR and Uch-L1 occurred in the cytoplasm. α₂AR agonist mediated activation of p44/42 MAP Kinase was drastically decreased in the presence of Uch-L1 indicating a functional relevance of this interaction. These findings may present a mechanism contributing to subtype-specific α₂AR trafficking and a potential pathway for the neuroprotective effects of α₂AR agonists.     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

Purification and partial characterisation of α2-antiplasmin and plasmin(ogen) from ostrich plasma

This study reports the isolation and partial characterisation of the ostrich serpin, α₂AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α₂AP was purified using lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a M_r of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α₂AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α₂AP, DFP and EACA. Ostrich plasminogen was highly purified after lysine–Sepharose chromatography and showed a M_r of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M_r of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively.     

Activation of phospholipase D by prostaglandin F2α in rat luteal cells and effects of inhibitors of arachidonic acid metabolism

In rat luteal cells labeled with (³H]oleic acid, PGF_2α-stimulated phospholipase D (PLD) activation was investigated. The PLD activity was detected by measuring the accumulation of [³H]phosphatidylethanol (PtdEt) in the presence of ethanol. PGF_2α stimulated PtdEt accumulation at concentrations of more than 100 nM in the presence of ethanol. However, PtdEt accumulation did not change in the absence of ethanol. PGF_2α (1 μM) increased PtdEt accumulation after 1 min, and the accumulation reached a plateau by 2–3 min. These results indicate that PGF_2α activates PLD in rat luteal cells. U-73122, a phospholipase C (PLC) inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, did not inhibit PGF_2α-stimulated [³H]PtdEt accumulation. These results suggest that PGF_2α-induced PLD activation is different from PLC-PKC systems. We reported previously that PGF_2α stimulated the release of arachidonic acid. The effects of indomethacin, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), inhibitors of arachidonic acid metabolism, on PGF_2α-stimulated PtdEt accumulation were examined. Pretreatment with indomethacin enhanced PGF_2α-induced PtdEt accumulation. In contrast, pretreatment with NDGA and ETYA inhibited PGF_2α-induced PtdEt accumulation. It is suggested that PGF_2α-stimulated PLD activation is mediated via lipoxygenase products.     

Genetic polymorphisms and plasma levels of transforming growth factor-beta(1) in Chinese patients with tuberculosis in Hong Kong

Susceptibility to tuberculosis (TB) may be affected by host genetic factors. Elevated levels of transforming growth factor-beta 1 (TGF-β₁) were found in plasma of patients with active TB compared with those of healthy contacts. To investigate the association of TGF-β₁ gene polymorphisms (C-509T and T869C) and plasma levels with the risk of TB in Hong Kong Chinese adults, a case-control study was carried out on 174 active TB patients and 174 healthy controls matched for age, gender and smoking. Blood samples from 180 blood donors served as another control group. Genotyping was carried out on genomic DNA using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma TGF-β₁ was measured by commercially available ELISA kit. We found no differences in the distribution of genotypes or alleles of TGF-β₁ gene polymorphisms at C-509T and T869C between patients and either group of healthy controls. Patients with TB had elevated plasma TGF-β₁ levels compared with healthy controls irrespective of their genotypes (p < 0.001). In conclusion, TGF-β₁ gene polymorphism at C-509T and T869C is not associated with TB susceptibility in Hong Kong Chinese adults, but elevated plasma TGF-β₁ levels suggests that this cytokine may play a role in the pathogenesis of tuberculosis.