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1.
Bone ; 29(4): 323-30, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11595614

ABSTRACT

Several members of the transforming growth factor-beta (TGF-beta) superfamily have been demonstrated to play regulatory roles in osteoblast differentiation and maturation, but the mechanisms by which they act on different cells at different developmental stages remain largely unknown. We studied the effects of TGF-beta1 and bone morphogenetic protein-2 (BMP-2) on the differentiation/maturation of osteoblasts using the murine cell lines MC3T3-E1 and C3H10T1/2. BMP-2 induced or enhanced the expression of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin (OC) in both cells. In contrast, TGF-beta1 was not only unable to induce these markers, but it dramatically inhibited BMP-2-mediated OC gene expression and ALP activity. In addition, TGF-beta1 inhibited the ability of BMP-2 to induce MC3T3-E1 mineralization. TGF-beta1 did not sensibly modify the increase of Osf2/Cbfa1 gene expression mediated by BMP-2, thus demonstrating that the inhibitory effect of TGF-beta1 on osteoblast differentiation/maturation mediated by BMP-2 was independent of Osf2/Cbfa1 gene expression. Finally, it is shown that TGF-beta1 does not affect BMP-2-induced Smad1 transcriptional activity in the mesenchymal pluripotent cells studied herein. Our data indicate that in vitro BMP-2 and TGF-beta1 exert opposite effects on osteoblast differentiation and maturation.


Subject(s)
DNA-Binding Proteins/pharmacology , Neoplasm Proteins , Osteoblasts/cytology , Osteoblasts/drug effects , Transforming Growth Factor beta/pharmacology , Alkaline Phosphatase/genetics , Animals , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Line , Core Binding Factor Alpha 1 Subunit , DNA-Binding Proteins/genetics , Enzyme Activation/drug effects , Gene Expression/drug effects , Mice , Osteoblasts/physiology , Osteocalcin/genetics , Signal Transduction/physiology , Smad Proteins , Smad1 Protein , Trans-Activators/genetics , Transcription Factors/genetics , Transforming Growth Factor beta1
2.
J Cell Sci ; 114(Pt 11): 2085-94, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11493644

ABSTRACT

The proteins of the hedgehog (Hh) family regulate various aspects of development. Recently, members of this family have been shown to regulate skeletal formation in vertebrates and to control both chondrocyte and osteoblast differentiation. In the present study, we analyzed the effect of Sonic hedgehog (Shh) on the osteoblastic and adipocytic commitment/differentiation. Recombinant N-terminal Shh (N-Shh) significantly increased the percentage of both the pluripotent mesenchymal cell lines C3H10T1/2 and ST2 and calvaria cells responding to bone morphogenetic protein 2 (BMP-2), in terms of osteoblast commitment as assessed by measuring alkaline phosphatase (ALP) activity. This synergistic effect was mediated, at least partly, through the positive modulation of the transcriptional output of BMPs via Smad signaling. Furthermore, N-Shh was found to abolish adipocytic differentiation of C3H10T1/2 cells both in the presence or absence of BMP-2. A short treatment with N-Shh was sufficient to dramatically reduce the levels of the adipocytic-related transcription factors C/EBPalpha and PPARgamma in both C3H10T1/2 and calvaria cell cultures. Given the inverse relationship between marrow adipocytes and osteoblasts with aging, agonists of the Hh signaling pathway might constitute potential drugs for preventing and/or treating osteopenic disorders.


Subject(s)
Adipocytes/cytology , Cell Differentiation , Cell Lineage , Mesoderm/cytology , Mesoderm/metabolism , Neoplasm Proteins , Osteoblasts/cytology , Trans-Activators/metabolism , Transforming Growth Factor beta , Adipocytes/drug effects , Adipocytes/enzymology , Adipocytes/metabolism , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/pharmacology , CCAAT-Enhancer-Binding Protein-alpha/genetics , Carrier Proteins , Cell Count , Cell Differentiation/drug effects , Cell Line , Cell Lineage/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Synergism , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Hedgehog Proteins , Mesoderm/drug effects , Mesoderm/enzymology , Mice , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteoblasts/metabolism , Osteocalcin/genetics , Promoter Regions, Genetic/genetics , Proteins/genetics , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Signal Transduction/drug effects , Skull/cytology , Smad Proteins , Trans-Activators/genetics , Trans-Activators/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger Protein GLI1
3.
Bone ; 28(5): 491-8, 2001 May.
Article in English | MEDLINE | ID: mdl-11344048

ABSTRACT

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.


Subject(s)
Bone Development/drug effects , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured/drug effects , MAP Kinase Signaling System/drug effects , Osteoblasts/drug effects , Stem Cells/drug effects , Transforming Growth Factor beta , Alkaline Phosphatase/genetics , Animals , Bone Development/physiology , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Cells, Cultured/cytology , Cells, Cultured/metabolism , Cricetinae , Enzyme Inhibitors/pharmacology , Female , Flavonoids , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Imidazoles/pharmacology , MAP Kinase Kinase 3 , MAP Kinase Signaling System/physiology , Mice , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Osteocalcin/genetics , Protein-Tyrosine Kinases/genetics , Pyridines/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Stem Cells/cytology , Stem Cells/metabolism , p38 Mitogen-Activated Protein Kinases
4.
Biochem Biophys Res Commun ; 271(3): 682-7, 2000 May 19.
Article in English | MEDLINE | ID: mdl-10814522

ABSTRACT

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Receptors, Cell Surface/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors , Transforming Growth Factor beta , Xenopus Proteins , Activin Receptors , Activin Receptors, Type I , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2 , Bone Morphogenetic Protein Receptors , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Cell Line , Cloning, Molecular , Gene Expression Regulation , Genes, Reporter , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Molecular Sequence Data , Nerve Growth Factors , Osteoblasts/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Smad Proteins , Smad4 Protein , Smad8 Protein
5.
Eur J Biochem ; 253(1): 76-83, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9578463

ABSTRACT

Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.


Subject(s)
Cysteine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Caspase 1 , Cell Line , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/isolation & purification , Humans , In Vitro Techniques , Insecta , Interleukin-1/metabolism , Kinetics , Oligopeptides/chemistry , Protein Precursors/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity
6.
Eur J Biochem ; 246(2): 394-400, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9208930

ABSTRACT

Interleukin-1beta converting enzyme (ICE) was the first identified member of a growing family of cysteine proteases that now includes ten mammalian homologs. Within this large family, two functional proteins, denoted TX and TY share 60% amino-acid identity with ICE in the mature protein and, together with ICE, constitute the ICE subfamily. The present study describes the identification of five new gene sequences, denoted S1-S5, closely related to ICE and TX and belonging to this subfamily. Sequences were identified using genomic Southern-blot analysis of human DNA with probes corresponding to ICE and TX exon 6. Using PCR amplification and cloning, the complete exon-6 sequence of these new genes was identified; three exhibit around 90% identity with Ice within exon 6, whereas the two others share about 70% identity with Ice. Examination of open reading frames and of amino acids essential for ICE activity indicate that none of these genes encodes for a functional protease. In conclusion, extensive analysis of the genes closely related to Ice shows that the Ice subfamily is constituted of eight members. Three of them encode for functional proteases (ICE, TX and TY) whereas the remaining members probably correspond to pseudogenes.


Subject(s)
Cysteine Endopeptidases/genetics , Amino Acid Sequence , Base Sequence , Caspase 1 , Cloning, Molecular , DNA, Complementary , Exons , Humans , Molecular Sequence Data , Open Reading Frames , Pseudogenes , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
7.
Apoptosis ; 2(2): 125-35, 1997.
Article in English | MEDLINE | ID: mdl-14646547

ABSTRACT

IL-1beta converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFalpha receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1beta processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.

8.
Eur J Biochem ; 236(1): 207-13, 1996 Feb 15.
Article in English | MEDLINE | ID: mdl-8617266

ABSTRACT

The present study describes the identification and molecular cloning of a new member of the interleukin-1 beta-converting enzyme (ICE) family denoted transcript Y (TY). TY is very closely related to both ICE (51% amino acid identity) and a protein named transcript X (TX) (75% amino acid identity) that we recently identified [Faucheu, C., Diu, A., Chan, A.W.E., Blanchet, A.-M., Miossec, C., Hervé, F.,Collard-Dutilleul, V., Gu, Y., Aldape, R., Lippke, J., Rocher, C., Su, M.S.-S., Livingston, D.J., Hercend, T. & Lalanne, J.-L. (1995) EMBO J. 14, 1914-1922]. The amino acids that are implicated in both the ICE catalytic site and in the PI aspartate-binding pocket are conserved in TY. Within the ICE gene family, TY belongs to a subfamily of proteins closely related to the prototype ICE protein. Using transfection experiments into mammalian cells, we demonstrate that TY has protease activity on its own precursor and that this activity is dependent on the presence of a cysteine residue at position 245. However, despite the close similarity between TY and ICE active sites, TY fails to process the interleukin-1 beta precursor. In addition, as already observed for ICE and TX, TY is able to induce apoptosis when overexpressed in COS cells. TY therefore represents a new member of the growing family of apoptosis-inducing ICE-related cysteine proteases.


Subject(s)
Apoptosis/genetics , Caspases , Cysteine Endopeptidases/genetics , Multigene Family , Amino Acid Sequence , Base Sequence , Caspase 1 , Caspases, Initiator , Cells, Cultured , Cloning, Molecular , Conserved Sequence , Cysteine Endopeptidases/classification , Cysteine Endopeptidases/metabolism , DNA, Complementary/genetics , Humans , Interleukin-1/metabolism , Leukocytes, Mononuclear/enzymology , Male , Molecular Sequence Data , Protein Precursors/metabolism , Recombinant Proteins/biosynthesis , Sequence Homology, Amino Acid , Substrate Specificity , Transfection
9.
EMBO J ; 14(9): 1914-22, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7743998

ABSTRACT

We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.


Subject(s)
Apoptosis/physiology , Caspases , Cysteine Endopeptidases/metabolism , Endopeptidases/metabolism , Amino Acid Sequence , Animals , Apoptosis/genetics , Base Sequence , Caspase 1 , Caspases, Initiator , Cell Line , Cloning, Molecular , Computer Simulation , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , DNA, Complementary/genetics , Endopeptidases/chemistry , Endopeptidases/genetics , Humans , Models, Molecular , Molecular Sequence Data , Multigene Family , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Transfection
10.
EMBO J ; 14(9): 1923-31, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7743999

ABSTRACT

Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.


Subject(s)
Cysteine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Caspase 1 , Cell Line , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , DNA, Complementary/genetics , Genetic Complementation Test , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Protein Processing, Post-Translational , Transfection
11.
Gene ; 98(2): 193-200, 1991 Feb 15.
Article in English | MEDLINE | ID: mdl-1673108

ABSTRACT

Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence. In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis. We constructed plasmids to test the synthesis of GPx in E. coli. These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E. coli and mammalian cells.


Subject(s)
DNA/genetics , Escherichia coli/genetics , Glutathione Peroxidase/genetics , Mutagenesis, Site-Directed , Animals , Base Sequence , Cloning, Molecular/methods , Codon/genetics , Cysteine/analogs & derivatives , DNA/isolation & purification , Gene Library , Genetic Vectors , Glutathione Peroxidase/biosynthesis , Liver/enzymology , Mice , Molecular Sequence Data , Oligonucleotide Probes , Plasmids , Poly A/genetics , Poly A/isolation & purification , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Restriction Mapping , Selenium , Selenocysteine
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