Mapping of the α4 subunit gene (GABRA4) to human chromosome 4 defines an α2—α4—β1—γ1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster

We demonstrated previously that an α₁—β₂—γ₂ gene cluster of the γ-aminobutyric acid (GABA_A) receptor is located on human chromosome 5q34–q35 and that an ancestral α—β—γ gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the α₄ gene (GABRA4) maps to human chromosome 4p14–q12, defining a cluster comprising the α₂, α₄, β₁, and γ₁ genes. The existence of an α₂—α₄—β₁—γ₁ cluster on chromosome 4 and an α₁—α₆—β₂—γ₂ cluster on chromosome 5 provides further evidence that the number of ancestral GABA_A receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the α gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of α subunit should be located on human chromosome 15q11–q13 within an α₅—α_x—β₃—γ₃ gene cluster at the locus for Angelman and Prader—Willi syndromes.     

TGF-β3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β-d-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β₃) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β₃ is added to TGF-β₃ null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-β₃, nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-β₃ is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β₃.     

Human G Protein γ11 and γ14 Subtypes Define a New Functional Subclass

The mammalian γ subunit family consists of a minimum of 12 members. Analysis of the amino acid sequence conservation suggests that the γ subunit family can be divided into three distinct subclasses. The division of the γ subunit family into these classes is based not only on amino acid homology, but also to some extent on functional similarities. In the present study, two new members of the γ subunit family, the γ₁₁ and γ₁₄ subunits, are identified and characterized in terms of their expression and function. The γ₁₁ and γ₁₄ subunits are most closely related to the γ₁ subunit and share similar biochemical properties, suggesting their inclusion in class I. However, despite their close phylogenetic relationship and similar biochemical properties, the γ₁, γ₁₁, and γ₁₄ subunits exhibit very distinct expression patterns, suggesting that class I should be further subdivided and that the signaling functions of each subgroup are distinct. In this regard, the γ₁₁ and γ₁₄ subunits represent a new subgroup of farnesylated γ subunits that are expressed outside the retina and have functions other than phototransduction.     

The α2δ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels

The auxiliary Ca_Vα₂δ-1 subunit is an important component of voltage-gated Ca²⁺ (Ca_V) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca_V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca_Vα₂δ-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca_V1.3 channels in HEK-293 cells. Co-expression of Ca_Vα₂δ-1 significantly increased macroscopic currents and conferred the Ca_V1.3α₁/Ca_Vβ₃ channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca_Vα₂δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of Ca_V1.3α₁/Ca_Vβ₃ currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant Ca_V1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca_Vα₂δ-1 auxiliary subunit may interact with Ca_V1.3 channels and regulate their functional expression.     

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.     

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-β₁ characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n = 12, 301 ± 41.22 g) was significantly higher than that of AI-R (n = 10, 204.8 ± 24.89 g) (p < 0.05). There were significantly lower E2 (p < 0.05) or higher P4 (p < 0.01) and TGF-β₁ (p < 0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-β₁ protein level compared to that of AI-R (p < 0.01). Interestingly, the TGF-β₁ protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.     

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.     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor β1 and β2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor β (TGF-β) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor β isoforms (TGF-β₁ and TGF-β₂) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGFβ was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-β isoforms.     

Integrin α5 during early development of Xenopus laevis

The full length sequence of the Xenopus integrin α₅ subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of α₅ mRNA occurs in the embryo. Furthermore, a variant form of the α₅ mRNA is expressed which encodes an integrin α₅ subunit with a truncated cytoplasmic domain. Integrin α₅ mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of α₅ mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the α₅ protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin α₅ subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of α subunit expression in early vertebrate embryos.     

Biphasic Effect of Transforming Growth Factor-β1 on in Vitro Angiogenesis

Although the existence of an increasing number of angiogenesis-regulating cytokines is well documented, the response elicited by combinations of these cytokines is largely unknown. Using an in vitro model in which microvascular endothelial cells can be induced to form capillary-like tubes within three-dimensional collagen or fibrin gels, we have investigated the effect of transforming growth factor-β₁ (TGF-β₁) on basic fibroblast growth factor (bFGF)-induced and vascular endothelial growth factor (VEGF)-induced angiogenesis. Endothelial cell invasion and capillary lumen formation were inhibited by TGF-β1 at relatively high concentrations (5-10 ng/ml), while lower concentrations (100 pg/ml-1 ng/ml) of TGF-β1 potentiated the effect of bFGF- and VEGF-induced invasion. The optimal potentiating effect was observed at 200-500 pg/ml TGF-β1. At invasion-potentiating doses of TGF-beta;1, lumen size in fibrin gels was markedly reduced compared to that in cultures treated with bFGF alone. These results show that TGF-β1 exerts a biphasic effect on bFGF- and VEGF-induced angiogenesis in vitro. Our studies support the notion that the nature of the angiogenic response elicited by a specific cytokine is contextual, i.e., depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.     

Activation of Integrin αVβ3 Regulates Cell Adhesion and Migration to Bone Sialoprotein

α_Vβ₃, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of α_Vβ₃, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for α_Vβ₃ in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of α_Vβ₃ activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn²⁺ markedly enhanced α_Vβ₃-dependent adhesion to BSP. α_Vβ₃-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that α_Vβ₃ activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by α_Vβ₃-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of α_Vβ₃ can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of α_Vβ₃ on neoplastic cells may contribute to tumor growth and metastatic potential.     

Uptake and degradation of α2-macroglobulin-protease complexes in human cells in culture

Serum components, present intracellularly in cultured human fibroblasts, were identified as α₂-macroglobulin (α₂M), albumin, α₁-trypsin inhibitor, hemopexin and transferrin, among others. These components were shown to be taken up from the culture medium. Kinetic analysis of the uptake of α₂M-trypsin complexes by the cells showed the uptake to be of a high affinity mechanism (K_M = 6 × 10⁻⁸ M α₂M in the medium), with a high rate of internalization (V_max=1.03 × 10⁶ molecules α₂M/cell and α₂M per hour). The intracellular degradation of α₂M is rapid, as judged by the half-life of 1.6 h. Virus-transformed or tumor-derived cell lines showed low or undetectable levels of α₂M. The possible physiological significance of the described phenomena is discussed in relation to the in vivo situation.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Retinoic acid down-regulates VPAC1 receptors and TGF-β3 but up-regulates TGF-β2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.     

Differential Modulation of Integrin Receptors and Extracellular Matrix Laminin by Transforming Growth Factor-β1 in Rat Alveolar Epithelial Cells

The transforming growth factors-β (TGFs-β) family of genes plays important roles in cell growth and differentiation in many cell types. TGFβ modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGFβ is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGFβ may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGFβ on modulation of laminin and its integrin receptors α₆β₁ and α₃β₁ in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGFβ₁ (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin β2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGFβ₁ induced dose-dependent increases in α₆ and β₁ mRNA levels. TGFβ₁ also increased the expression of laminin β2 chain mRNA at 12-16 h poststimulation. In contrast, TGFβ decreased α₃ mRNA expression. Immunoprecipitation studies of TGFβ₁-treated cells showed increased surface expression of both α₆ and β₁ protein while surface expression of the α₃ integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGFβ₁ may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.     

A simple method for preparation of valency hybrid hemoglobins, (αβ)2 and (αβ)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.