TGFβ2 in Corneal Morphogenesis during Mouse Embryonic Development

To examine the roles of TGFβ isoforms on corneal morphogenesis, the eyes of mice that lack TGFβs were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb^−/− mice, only Tgfb2^−/− mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2^−/− mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFβ2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2^−/− mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2^−/− mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice

The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alpha v beta6 and alpha v beta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system.     

Knocking out the regulatory beta subunit of protein kinase CK2 in mice: Gene dosage effects in ES cells and embryos

Knocking out the regulatory β subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2β (CK2β^+/−) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2β^+/− inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2β^+/− ES (Embryonic Stem) cells express a considerably lower level of CK2β than wild-type ES cells, whereas the level of CK2β in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2β levels during development in the majority of, but not in all, cases (Mol Cell Biol {23:} 908–915, 2003).In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2β^+/− ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2β^+/− embryos. No correlation between CK2β levels in normal looking and retarded CK2β^+/− embryos were found. However, a different post-translational form of CK2β protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2β^+/− cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2β.     

MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Lipid radical generation in polar (Laternula elliptica) and temperate (Mya arenaria) bivalves

Lipid peroxidation in Laternula elliptica was assessed by detecting lipid radicals by electronic paramagnetic resonance. The values were compared with data from the temperate mud clam Mya arenaria. Lipid radical content was higher in the Antarctic bivalve than in the temperate mud clam, even within the range of its habitat temperature. The rate of generation of lipid radicals was affected by the iron content in the samples. The iron content in individual samples of digestive glands in L. elliptica ranged from 3 to 6 nmol g⁻¹ fresh weight (fwt) and in M. arenaria from 0.6 to 2.7 nmol g⁻¹ fwt. Arrhenius plots, developed from the rates obtained in the presence of 25 μM iron, showed no significant differences between the activation energy calculated for digestive glands of L. elliptica and M. arenaria. The Fe³⁺ reduction rate in L. elliptica was higher than in M. arenaria (4.7±0.9 vs. 1.8±0.4 nmol mg⁻¹ protein min⁻¹, respectively). L. elliptica had a higher content of α-tocopherol and β-carotene than M. arenaria. Our data suggest that increased lipid radical content in the membranes of cold-adapted organisms could be related to iron content.     

Microfibril-associated Glycoprotein 2 (MAGP2) Loss of Function Has Pleiotropic Effects in Vivo

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5^−/− mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2^−/−;Mfap5^−/−) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFβ1, TGFβ2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling.     

Localization of αυβ6 Integrin-TGF-β1/Smad3, mTOR and PPARγ in Experimental Colorectal Fibrosis

A simultaneous action of several pro-fibrotic mediators appears relevant in the development of fibrosis. There are evidences that transforming growth factor-β (TGF-β)/Smad3 pathway forms with αvβ6 integrin, mammalian target of Rapamycin (mTOR) and peroxisome proliferator-activated receptor-γ (PPARγ) a complex signalling network with extensive crosstalk and strong effects on fibrosis development. The present study evaluated the expression of TGFβ, Smad3, αvβ6 integrin, mTOR and PPARγ in 2, 4, 6-trinitrobenzenesulphonic acid (TNBS)-induced colorectal fibrosis in Smad3 wild-type (WT) and null mice. Smad3 WT mice treated with TNBS developed a marked colorectal fibrosis and showed a concomitant up-regulation of TGFβ, Smad3, αvβ6 and mTOR and a reduction of PPARγ expression. On the other hand, Smad3 Null mice similarly treated with TNBS did not develop fibrosis and showed a very low or even absent expression of TGFβ, Smad3, αvβ6 and mTOR and a marked over-expression of PPARγ. At the same time the expression of α-smooth muscle actin (a marker of activated myofibroblasts), collagen I-III and connective tissue growth factor (a downstream effector of TGFβ/Smad3-induced extracellular matrix proteins) were up-regulated in Smad3 WT mice treated with TNBS compared to Null TNBS-treated mice. These preliminary results suggest a possible interaction between these pro-fibrotic molecules in the development of intestinal fibrosis.Key words: intestinal fibrosis, integrins, TGF-β, SMAD, mTOR, PPAR, IBD     

Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells was investigated. As zinc compounds, we used zinc sulfate, AHZ, di(N-acetyl-β-alanyl-l-histidinato)zinc (AAHZ), and di(histidino)zinc (HZ). Cells were cultured for 72 h in the presence of zinc compounds (10⁻⁸–10⁻⁵M). The effect of AHZ (10⁻⁷ and 10⁻⁶M) to increase protein and deoxyribonucleic acid (DNA) contents in the cells was the greatest in comparison with those of other zinc compounds. Zinc sulfate and HZ at 10⁻⁷M did not have an effect on the cellular protein content. AHZ (10⁻⁶M) had a potent effect on cell proliferation, although zinc sulfate (10⁻⁶M) had no effect. β-Alanyl-l-histidine (10⁻⁶ and 10⁻⁵M) did not have an appreciable effect on the cells. Those effects of AHZ (10⁻⁶M) on osteoblastic cells were completely abolished by the presence of cycloheximide (10⁻⁶M). AHZ (10⁻⁸–10⁻⁵M) directly activated [³H]leucyl-tRNA synthetase in the cell homogenate, whereas the effect of zinc sulfate was seen at 10⁻⁶ and 10⁻⁵M. The present study suggests that the chemical form of zinc-chelating β-alanyl-l-histidine (AHZ) can reveal a potent anabolic effect on osteoblastic cells, and that AHZ directly stimulates protein synthesis.     

IGF-II Enhances Trichostatin A-Induced TGFβ1 and p21 Expression in Hep3B Cells

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFβ1. Latent TGFβ1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFβ1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFβ1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFβ1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFβ1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.     

Disrupted Sperm Function and Fertilin β Processing in Mice Deficient in the Inositol Polyphosphate 5-Phosphatase Inpp5b

Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b^−/− males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin β, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b^−/− animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b^−/− mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.     

TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development

Transforming growth factor-β (TGF-β) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-β signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2^fl/fl;Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-β signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2^fl/fl;Wnt1-Cre mice. Moreover, exogenous TGF-β can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2^fl/fl;Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2^fl/fl;Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-β signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-β signaling.     

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

Microfluorometric analysis of Cl permeability and its relation to oscillatory Ca signalling in glucose-stimulated pancreatic β-cells

The cytoplasmic concentrations of Cl⁻([Cl⁻]_i) and Ca²⁺ ([Ca²⁺]_i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl⁻]_i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl⁻ into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl⁻]_i either in the absence or presence of furosemide inhibition of Na⁺, K⁺, 2 Cl⁻ co-transport. Glucose-induced oscillations of [Ca²⁺]_i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H₂DIDS). A similar effect was noted when replacing 25% of extracellular Cl⁻ with the more easily permeating anions SCN⁻, I⁻, NO₃⁻ or Br⁻. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl⁻ permeability and that the oscillatory Ca²⁺ signalling is critically dependent on transmembrane Cl⁻ fluxes.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.