WFIKKN1 and WFIKKN2: “Companion” proteins regulating TGFB activity

The WFIKKN (WAP, Follistatin/kazal, Immunoglobulin, Kunitz and Netrin domain-containing) protein family is composed of two multidomain proteins: WFIKKN1 and WFIKKN2. They were formed by domain shuffling and are likely present in deuterostoms. The WFIKKN (also called GASP) proteins are well known for their function in muscle and skeletal tissues, namely, inhibition of certain members of the transforming growth factor beta (TGFB) superfamily such as myostatin (MSTN) and growth and differentiation factor 11 (GDF11). However, the role of the WFIKKN proteins in other tissues is still poorly understood in spite of evidence suggesting possible action in the inner ear, brain and reproduction. Further, several recent studies based on next generation technologies revealed differential expression of WFIKKN1 and WFIKKN2 in various tissues suggesting that their function is not limited to MSTN and GDF11 inhibition in musculoskeletal tissue. In this review, we summarize current knowledge about the WFIKKN proteins and propose that they are “companion” proteins for various growth factors by providing localized and sustained presentation of TGFB proteins to their respective receptors, thus regulating the balance between the activation of Smad and non-Smad pathways by TGFB.     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor (TGF), and transforming growth factor 1 (TGF1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGF improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGF. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGF1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGF1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGF, and TGF1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 145–150.Original Russian Text Copyright © 2005 by Penkov, Platonov, Dimitrov, Mironova, Konyukhov.     

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.     

β-Linked disaccharides stimulate,but do not act as primer for,β(1–3)glucan synthase activity ofNeurospora crassa

-Linked disaccharides (laminaribiose and cellobiose) stimulated(1–3)glucan synthase activity ofNeurospora crassa by reducing the K_{m app} for the substrate while not changing the V_max. Laminaribiose and cellobiose werelinear activators with a K_{a app} of 0.32 mM and K_{a app} of 1.7 mM, respectively. Laminaribiose was not found to be incorporated into product, i.e., did not act as a primer covalently bound to product.     

Expression of TGF-β1 and β3 but not apoptosis factors relates to flow-induced aortic enlargement

Background  

Cell proliferation and apoptosis are both involved in arterial wall remodeling. Increase in blood flow induces arterial enlargement. The molecular basis of flow-induced remodeling in large elastic arteries is largely unknown.     

Polymorphisms of VEGF,TGFβ1, TGFβR2 and conotruncal heart defects in a Chinese population

Genetic variants may determine susceptibility of congenital heart disease (CHD). To evaluate the impact of transforming growth factor-β1 (TGFβ1), TGFβ receptor II (TGFβR2) and vascular endothelial growth factor (VEGF) polymorphisms on conotruncal heart defects susceptibility, we genotyped six functional polymorphisms TGFβ1 rs1800469 C>T, TGFβR2 rs3087465 G>A, VEGF ?2578C>A, ?1498T>C, ?634G>C and +936C>T in a hospital based case–control study of 244 conotruncal heart defects cases and 136 non-CHD controls in a Chinese population. Logistic regression analyses revealed that if the TGFβ1 rs1800469 CC homozygote genotype was used as the reference group, subjects carrying the CT variant heterozygote had a significant 0.48-fold decreased risk of conotruncal heart defects [odds ratio (OR) = 0.52; 95 % confidence interval (CI) = 0.30–0.88], subjects carrying the TT variant homozygote had a significant 0.47-fold decreased risk of conotruncal heart defects (OR 0.53; 95 % CI 0.28–1.00). In stratification analyses, the TGFβ1 rs1800469 C>T genotype was associated with a decreased risk for tetralogy of fallot in homozygote comparisons (OR 0.47; 95 % CI 0.22–0.99), a decreased risk for transposition of great artery in the dominant genetic model (OR 0.49; 95 % CI 0.28–0.87) and heterozygote comparisons (OR 0.45; 95 % CI 0.24–0.83). Our findings suggest that TGFβ1 rs1800469 C>T polymorphism was significantly associated with decreased risk of conotruncal heart defects. TGFβR2 rs3087465 G>A, VEGF ?2578C>A, ?1498T>C, ?634G>C and +936C>T polymorphisms may not play a role in the susceptibility of conotruncal heart defects.     

Transforming growth factor-β1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)-β1, placenta growth factor (PlGF)-1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF-receptor (VEGFR)-mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF-β1-induced monocyte migration is fully functional. Therefore, we hypothesize that TGF-β antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF-β1, while PlGF-1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF-β receptor in monocytes under high-glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down-regulation of TGF-β receptor expression in vitro or by functional blocking of TGF-β signalling using either a TGF-β receptor kinase inhibitor or a TGF-β neutralizing antibody. Our data demonstrate that both T2DM and high-glucose potentiate the TGF-β pathway. TGF-β signalling impairs VEGFR-mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.     

β2-Agonists upregulate PDE4 mRNA but not protein or activity in human airway smooth muscle cells from asthmatic and nonasthmatic volunteers

β(2)-Adrenergic receptor (β2AR) agonists induce airway relaxation via cAMP. Phosphodiesterase (PDE)s degrade and regulate cAMP, and in airway smooth muscle (ASM) cells PDE4D degrades cAMP. Long-acting β(2)-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budesonide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)-β(1), formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with β2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis.     

Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.     

TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.     

The TGFβ-induced phosphorylation and activation of p38 mitogen-activated protein kinase is mediated by MAP3K4 and MAP3K10 but not TAK1

The signalling pathways downstream of the transforming growth factor beta (TGFβ) family of cytokines play critical roles in all aspects of cellular homeostasis. The phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in TGFβ-induced epithelial-to-mesenchymal transition and apoptosis. The precise molecular mechanisms by which TGFβ cytokines induce the phosphorylation and activation of p38 MAPK are unclear. In this study, I demonstrate that TGFβ-activated kinase 1 (TAK1/MAP3K7) does not play a role in the TGFβ-induced phosphorylation and activation of p38 MAPK in MEFs and HaCaT keratinocytes. Instead, RNAi-mediated depletion of MAP3K4 and MAP3K10 results in the inhibition of the TGFβ-induced p38 MAPK phosphorylation. Furthermore, the depletion of MAP3K10 from cells homozygously knocked-in with a catalytically inactive mutant of MAP3K4 completely abolishes the TGFβ-induced phosphorylation of p38 MAPK, implying that among MAP3Ks, MAP3K4 and MAP3K10 are sufficient for mediating the TGFβ-induced activation of p38 MAPK.     

Activin A and transforming growth factor-β stimulate heart formation in axolotls but do not rescue cardiac lethal mutants

In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-1 or 2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-1 and 5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesodermal explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor- superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.F.J. Mangiacapra and M.E. Fransen contributed equally to this work     

Synthesis,structure and anti-acetylcholinesterase activity of 4β-methoxymethyl-4β-demethyl territrem B

4β-Methoxymethyl-4β-demethyl territrem B [5] was synthesized from 4β-hydroxymethyl-β-demethyl territrem B [4] by treatment with dimethyl sulfate in methanolic NaOH. The structure of 5 was elucidated by uv, nmr and mass spectra. The IC50 of 5 on acetylcholinesterase (AChE) was 6.30 × 10-5 M, which indicated 0.4% of the anti-AChE activity of territrem B [2]. This revised version was published online in August 2006 with corrections to the Cover Date.     

How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.