TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart.
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Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM. (+info)
Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages.
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Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells. (+info)
Plasma from human mothers of fetuses with severe arthrogryposis multiplex congenita causes deformities in mice.
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Arthrogryposis multiplex congenita (AMC) is characterized by fixed joint contractures and other deformities, sometimes resulting in fetal death. The cause is unknown in most cases, but some women with fetuses affected by severe AMC have serum antibodies that inhibit fetal acetylcholine receptor (AChR) function, and antibodies to fetal antigens might play a pathogenic role in other congenital disorders. To investigate this possibility, we have established a model by injecting pregnant mice with plasma from four anti-AChR antibody-positive women whose fetuses had severe AMC. We found that human antibodies can be transferred efficiently to the mouse fetus during the last few days of fetal life. Many of the fetuses of dams injected with AMC maternal plasmas or Ig were stillborn and showed fixed joints and other deformities. Moreover, similar changes were found in mice after injection of a serum from one anti-AChR antibody-negative mother who had had four AMC fetuses. Thus, we have confirmed the role of maternal antibodies in cases of AMC associated with maternal anti-AChR, and we have demonstrated the existence of pathogenic maternal factors in one other case. Importantly, this approach can be used to look at the effects of other maternal human antibodies on development of the fetus. (+info)
Enhancement of cell adhesion and spreading by a cartilage-specific noncollagenous protein, cartilage matrix protein (CMP/Matrilin-1), via integrin alpha1beta1.
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Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage. (+info)
Cyclic stretch regulates autocrine IGF-I in vascular smooth muscle cells: implications in vascular hyperplasia.
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Vascular smooth muscle cells (VSMC) subjected to acute or chronic stretch display enhanced growth rates in vitro and in vivo. Clinical examples of vascular hyperplasia (e.g., systolic hypertension and postinjury restenosis) suggest that local insulin-like growth factor I (IGF-I) expression is enhanced. Therefore, we investigated the role of in vitro cyclic stretch on rat VSMC IGF-I secretion and cellular growth. In serum-free medium, cyclic stretch (1 Hz at 120% resting length for 48 h) stimulated thymidine incorporation approximately 40% above that seen in nonstretched cells. Graded stretch magnitude (100-125% resting length) yielded graded increases in VSMC growth. Exogenous IGF-I increased growth of serum-starved, nonstretched VSMC in a dose-dependent manner, with maximal growth seen with 10(-7) M. IGF-I secretion from stretched cells was 20- to 30-fold greater than from those cells cultured in a static environment. Stretch-induced increases in growth were completely blocked on addition of anti-IGF-I and partially blocked with platelet-derived growth factor (PDGF) antibodies and with a tyrosine kinase inhibitor (tyrphostin-1). Finally, blockade of stretch-activated cation channels with GdCl3 profoundly inhibited stretch-induced growth. We conclude that stretch increases VSMC IGF-I secretion and that such autocrine IGF-I is required for stretch-induced growth. PDGF and stretch-sensitive cation channels are likely additional components of a complex pathway that regulates stretch-induced VSMC seen in systolic hypertension and postinjury restenosis. (+info)
Muscle LIM protein: expressed in slow muscle and induced in fast muscle by enhanced contractile activity.
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To identify early changes in gene expression during the fast-to-slow transition induced by chronic low-frequency stimulation, total RNA was extracted from 12-h-stimulated tibialis anterior (TA) muscles of rats and amplified by differential display RT-PCR. Among the signals of differentially expressed mRNAs, a cDNA approximately 300 bp in length, which was almost undetectable in control TA muscles but prominent in stimulated TA and normal soleus muscles, was identified. This cDNA was cloned and identified as corresponding to the mRNA of the muscle LIM protein (MLP). Its differential expression in control, stimulated TA, and soleus muscles was verified by Northern blotting. Antibodies against MLP were used to identify by immunoblot analysis a protein of 22 kDa, the predicted molecular mass of MLP. Immunohistochemistry revealed strong reactivity for MLP in all fibers of normal soleus muscle and faint staining of some type IIA and type I fibers in control TA muscle. These fibers increased in number and staining intensity in 4-day-stimulated TA muscle. MLP thus seems to play an essential role during the rearrangement of cytoskeletal and/or myofibrillar structures in transforming adult muscle fibers. (+info)
Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas.
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CPP32/apopain (Caspase-3), a protease of the Ced-3/ICE family, is a central mediator in the apoptosis induced by TNF or anti-Fas. In this study we demonstrate that wortmannin, an inhibitor of PI-3K, enhances the activation of CPP32 (Caspase-3) and DNA fragmentation in TNF-treated U937 cells and anti-Fas-treated Jurkat cells. Caspase-3-like activity, Ac-DEVD-MCA cleavage activity, is enhanced by wortmannin in the range of the concentration (1 - 100 nM) specifically inhibiting PI-3K. LY294002, another PI-3K inhibitor, also enhances Caspase-3-like activity, but inhibitors for myosin light chain kinase and calmodulin dependent kinase do not have any effect on the Caspase-3-like activity. Wortmannin (1 - 100 nM) enhances the processing of Caspase-3 (32K) into active form (17K) in TNF- or anti-Fas-treated cells, but not in untreated cells. These observations suggest that inhibition of PI-3K induces the activation of processing enzyme of Caspase-3 or increases the susceptibility of Caspase-3 to the processing enzyme. PI-3K seems to protect the cells from apoptosis by suppressing the activation of Caspase-3. (+info)
JNK/SAPK activity contributes to TRAIL-induced apoptosis.
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We report here that JNK/SAPKs are activated by TRAIL in parallel to induction of apoptosis in human T and B cell lines. Death signaling as well as JNK/SAPK activation by TRAIL in these cells is FADD- and caspase-dependent since dominant-negative FADD or the caspase inhibitor zVAD prevented both, apoptosis and JNK/SAPK activity. JNK/SAPK activity in response to triggering of CD95 by an agonistic antibody (alphaAPO-1) was also diminished by dominant-negative FADD or zVAD. Correspondingly, a cell line resistant to alphaAPO-1-induced death exhibited crossresistance to TRAIL-induced apoptosis and did not upregulate JNK/SAPK activity in response to TRAIL or alphaAPO-1. Inhibition of JNK/SAPK activity, by stably transfecting cells with a dominant-negative JNKK-MKK4 construct, reduced apoptosis in response to TRAIL or alphaAPO-1. Therefore, activation of JNK/SAPKs by TRAIL or alphaAPO-1 occurs downstream of FADD and caspases and contributes to apoptosis in human lymphoid cell lines. (+info)