Histone demethylase Kdm4b functions as a co-factor of C/EBPß to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes.

CCAAT/enhancer-binding protein (C/EBP) ß is required for both mitotic clonal expansion (MCE) and terminal adipocyte differentiation of 3T3-L1 preadipocytes. Although the role of C/EBPß in terminal adipocyte differentiation is well defined, its mechanism of action during MCE is not. In this report, histone demethylase Kdm4b, as well as cell cycle genes Cdc45l (cell division cycle 45 homolog), Mcm3 (mini-chromosome maintenance complex component 3), Gins1 (GINS complex subunit 1) and Cdc25c (cell division cycle 25 homolog c), were identified as potential C/EBPß target genes during MCE by utilizing promoter-wide chromatin immunoprecipitation (ChIP)-on-chip analysis combined with gene expression microarrays. The expression of Kdm4b is induced during MCE and its induction is dependent on C/EBPß. ChIP, Electrophoretic Mobility Shift Assay (EMSA) and luciferase assay confirmed that the promoter of Kdm4b is bound and activated by C/EBPß. Knockdown of Kdm4b impaired MCE. Furthermore, Kdm4b interacted with C/EBPß and was recruited to the promoters of C/EBPß-regulated cell cycle genes, including Cdc45l, Mcm3, Gins1, and Cdc25c, demethylated H3K9me3 and activated their transcription. These findings suggest a novel feed forward mechanism involving a DNA binding transcription factor (C/EBPß) and a chromatin regulator (Kdm4b) in the regulation of MCE by controlling cell cycle gene expression.

Distinct functional domains of TGF-beta bind receptors on endothelial cells.

Transforming growth factor-beta (TGF-beta) is a multi-functional regulator of cell growth and differentiation. Three distinct isoforms of TGF-beta exist having similar, but not identical actions. TGF-beta 1, but not TGF-beta 2, binds to T beta RII and also to endoglin, a cell surface protein abundant on endothelial cells. In contrast, the affinity constant of TGF-beta 2 for alpha 2-macroglobulin is 10-fold greater than that of TGF-beta 1. TGF-beta 2 also binds better than TGF-beta 1 to a glycosyl phosphatidylinositol (GPI)-linked binding protein expressed on vascular endothelial cells. Using chimeric TGF-beta molecules, in which selected regions of TGF-beta 1 have been exchanged for the corresponding region of TGF-beta 2, we demonstrate here that amino acids 92-95 or 94-98 of TGF-beta determine isoform specific binding to endoglin. In contrast, exchange of only amino acids 95 and 98 did not alter TGF-beta specificity. Isoform specific binding to a GPI-linked protein on EJG endothelial cells was modulated by a region containing amino acids 40-68, although exchange of only amino acids 40-47 did not confer isoform specific binding. Significantly, the 92-98 region also modulates binding of TGF-beta to the type II receptor whereas isoform specific binding to alpha 2-macroglobulin requires concerted exchange of amino acids 45 and 47. Taken together, these results show that at least three different functional domains are important modulators of TGF-beta interaction with binding proteins and receptors.

Mutational analysis of a transforming growth factor-beta receptor binding site.

Transforming growth factor-beta s (TGF-beta 1, -beta 2, -beta 3) are important regulators of cell growth and differentiation which share approximately 70% identical amino acids. Using LS513 colorectal cells, which are growth inhibited by TGF-beta 1 (ED50 of 100 pM), but are refractory to TGF-beta 2 (ED50 of 50,000 to 100,000 pM), we have determined that amino acids 92-98 of TGF-beta specify growth inhibition. The chimeric protein TGF-beta 1/beta 2(92-98), in which amino acids 92-98 of TGF-beta 1 were exchanged for the corresponding amino acids of TGF-beta 2, was indistinguishable from TGF-beta 2 at inhibiting growth of LS513 cells. In contrast, both TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) inhibited the growth of LS513 cells with an ED50 of approximately 1000 pM. TGF-beta 1/beta 2(95-98), in which amino acids 95-98 of TGF-beta 1 have been replaced with the corresponding amino acids of TGF-beta 2, had full activity and was indistinguishable from TGF-beta 1. Receptor cross-linking experiments demonstrated that binding of the chimeras to the type I and type II receptors of LS513 cells was consistent with their biological activity. TGF-beta 1/beta 2(95-98), TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) were each similar to TGF-beta 2 in that they failed to bind to the soluble Type II receptor in a solid-phase assay. These results demonstrate that amino acids 92-98 are involved in the interaction between TGF-beta and its signaling receptors and they show that modest changes within this region can substantially alter biological response.

Binding affinity of transforming growth factor-beta for its type II receptor is determined by the C-terminal region of the molecule.

Transforming growth factor-beta (TGF-beta) isoforms have differential binding affinities for the TGF-beta type II receptor (TbetaRII). In most cells, TGF-beta1 and TGF-beta3 bind to TbetaRII with much higher affinity than TGF-beta2. Here, we report an analysis of the effect of TGF-beta structure on its binding to TbetaRII by using TGF-beta mutants with domain deletions, amino acid replacements, and isoform chimeras. Examination of the binding of TGF-beta mutants to the recombinant extracellular domain of TbetaRII by a solid-phase TGF-beta/TbetaRII assay demonstrated that only those TGF-beta mutants containing the C terminus of TGF-beta1 (TGF-beta1-(Delta69-73), TGF-beta1-(Trp71), and TGF-beta2/beta1-(83-112)) bind with high affinity to TbetaRII, similar to native TGF-beta1. Moreover, replacement of only 6 amino acids in the C terminus of TGF-beta1 with the corresponding sequence of TGF-beta2 (TGF-beta1/beta2-(91-96)) completely eliminated the high affinity binding of TGF-beta1. Proliferation of fetal bovine heart endothelial (FBHE) cells was inhibited to a similar degree by all of the TGF-beta mutants. However, recombinant soluble TbetaRII blocked the inhibition of FBHE cell proliferation induced by TGF-beta mutants retaining the C terminus of TGF-beta1, consistent with the high binding affinity between these TGF-beta molecules and TbetaRII. It was further confirmed that the TGF-beta2 mutant with its C terminus replaced by that of TGF-beta1 (TGF-beta2/beta1-(83-112)) competed as effectively as TGF-beta1 with 125I-TGF-beta1 for binding to membrane TbetaRI and TbetaRII on FBHE cells. These observations clearly indicate that the domain in TGF-beta1 responsible for its high affinity binding to TbetaRII, both the soluble and membrane-bound forms, is located at C terminus of the molecule.

Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2.

The three-dimensional solution structure of human transforming growth factor beta 1 (TGF-beta 1) has been determined using multinuclear magnetic resonance spectroscopy and a hybrid distance geometry/ simulated annealing algorithm. It represents one of the first examples of a mammalian protein structure that has been solved by isotopic labeling of the protein in a eukaryotic cell line and multinuclear NMR spectroscopy. The solution structure of the 25 kDa disulfide-linked TGF-beta 1 homodimer was calculated from over 3200 distance and dihedral angle restraints. The final ensemble of 33 accepted structures had no NOE or dihedral angle violations greater than 0.30 A and 5.0 degrees, respectively. The RMSD of backbone atoms for the ensemble of 33 structures relative to their mean structure was 1.1 A when all residues were used in the alignment and 0.7 A when loop regions were omitted. The solution structure of TGF-beta 1 follows two independently determined crystal structures of TGF-beta 2 (Daopin et al., 1992, 1993; Schlunegger & Grütter, 1992, 1993), providing the first opportunity to examine structural differences between the two isoforms at the molecular level. Although the structures are very similar, with an RMSD in backbone atom positions of 1.4 A when loop regions are omitted in the alignment and 1.9 A when all residues are considered, there are several notable differences in structure and flexibility which may be related to function. The clearest example of these is in the beta-turn from residues 69-72: the turn type found in the solution structure of TGF-beta 1 falls into the category of type II, whereas that present in the X-ray crystal structure of TGF-beta 2 is more consistent with a type I turn conformation. This may be of functional significance as studies using TGF-beta chimeras and deletion mutants indicate that this portion of the molecule may be important in receptor binding.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Transforming growth factor beta isoform 2-specific high affinity binding to native alpha 2-macroglobulin. Chimeras identify a sequence that determines affinity for native but not activated alpha 2-macroglobulin.

Transforming growth factor beta 2 (TGF-beta 2) is less potent than TGF-beta 1 in some endothelial cell proliferation assays due to the greater tendency of TGF-beta 2 to bind alpha 2-macroglobulin (alpha 2M). Substitution of TGF-beta 1 residues 40-47 into the TGF-beta 2 sequence yields a chimeric molecule that, like TGF-beta 1, expresses activity that is not substantially affected by serum alpha 2M (Burmester, J. K., Qian, S. W., Roberts, A. B., Huang, A., Amatayakul-Chantler, S., Suardet, L., Odartchenko, N., Madri, J. A., and Sporn, M. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8628-8632). In this investigation, we studied the binding of TGF-beta chimeras, which contain TGF-beta 1 residues 40-47, to both major conformations of human alpha 2M under apparent equilibrium conditions. Native alpha 2M, the primary form of this protein in serum, bound TGF-beta 2/beta 1 (40-82) and TGF-beta 2/beta 1 (40-47) with low affinity. The apparent KD values for the two chimeras and native alpha 2M were 310 and 330 nM, respectively. These values were much higher than the KD determined for TGF-beta 2 and native alpha 2M (11 nM) and equivalent to the KD determined for TGF-beta 1 and native alpha 2M. By contrast, both TGF-beta chimeras bound alpha 2M-methylamine, an altered conformation of alpha 2M, with high affinity (16 and 19 nM), which is characteristic of TGF-beta 2 and not TGF-beta 1. Fetal bovine heart endothelial cell DNA synthesis was inhibited to a similar degree by TGF-beta 1, TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) in the presence of dilute (0.2%) fetal bovine serum. When 0.07 microM alpha 2M-methylamine was added, the activities of TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) were significantly counteracted while the activity of TGF-beta 1 was unchanged, as would be predicted by the equilibrium binding analyses. These studies indicate that the TGF-beta structural elements, which mediate binding to native alpha 2M and conformationally transformed alpha 2M, are not equivalent. Residues 40-47 are critical in determining affinity for native alpha 2M but are less important in determining affinity for alpha 2M-methylamine.

[Ser77]transforming growth factor-beta 1. Selective biological activity and receptor binding in mink lung epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) is a homodimeric protein stabilized by a single disulfide bridge between Cys77 on the respective monomers and two paired complementary hydrophobic interfaces between the two subunits. A TGF-beta 1 mutant with Cys77 replaced by serine has been expressed in stably transfected Chinese hamster ovary cells and purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirms that the sole interchain disulfide bond in TGF-beta 1 has been eliminated. It is 20% as potent as native TGF-beta 1 in the induction of plasminogen activator inhibitor-1 promoter expression in mink lung epithelial cells (Mv1Lu), although it is less than 1% as potent as native TGF-beta 1 in inhibition of growth in the same cell line. The mutant acts as a full agonist in both bioassays. [Ser77]TGF-beta 1 binds to soluble type II receptors and competes with native TGF-beta 1 in sandwich-enzyme-linked immunosorbent assays; however, in Mv1Lu cells, the mutant shows preferential cross-linking to type I rather than type II receptors. [Ser77]TGF-beta 1 is a useful tool for understanding the different ligand-receptor complexes and numerous biological activities of this multifunctional cytokine.

Characterization of mutated transforming growth factor-beta s which possess unique biological properties.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of distinct functional domains of transforming growth factor beta.

Three distinct isoforms of transforming growth factor beta (TGF-beta) are expressed in mammalian cells. Although many cells respond equivalently to all three isoforms, certain cells respond selectively. Using chimeric proteins in which selected regions of the different isoforms were interchanged, we have identified two distinct functional domains of TGF-beta involved in determining the biological potencies and functions of the molecule. The first domain is important for determining whether TGF-beta can be sequestered by alpha 2-macroglobulin. By replacing aa 45 and 47 of TGF-beta 2 with the corresponding amino acids of TGF-beta 1, sequestration of the TGF-beta molecule by alpha 2-macroglobulin was markedly reduced. The second domain is functionally different from the alpha 2-macroglobulin sequestration site and is important for determining the potency of TGF-beta to inhibit growth of the LS513 human colorectal cancer cell line. Neither the TGF-beta 2/beta 1-(40-47) replacement construct nor a chimera containing aa 1-39 of TGF-beta 2, aa 40-82 of TGF-beta 1, and aa 83-112 of TGF-beta 2 was equivalent to TGF-beta 1 in inhibiting growth of LS513 cells. This fact suggests that additional amino acids outside of the aa 40-82 region are required to specify TGF-beta 1 activity with these cells.

Identification of a structural domain that distinguishes the actions of the type 1 and 2 isoforms of transforming growth factor beta on endothelial cells.

A chimeric transforming growth factor beta (TGF-beta) molecule has been synthesized to map the amino acids responsible for the substantially greater activity of TGF-beta 1 than TGF-beta 2 on growth and migration of endothelial cells. This chimera consists of a dimer of a monomeric unit composed of amino acids 1-39 of TGF-beta 2, 40-82 of TGF-beta 1, and 83-112 of TGF-beta 2. Structural identity of the purified recombinant protein has been confirmed by immunoblotting and NH2-terminal sequencing. The biological potency of the TGF-beta 2-1-2 chimera was equal to that of TGF-beta 1 in inhibition of growth of both fetal bovine heart endothelial cells and rat epididymal fat pad microvascular endothelial cells. Similarly, the TGF-beta 2-1-2 chimera was nearly equivalent to TGF-beta 1 and at least 10-fold more active than TGF-beta 2 in inhibiting migration of bovine aortic endothelial cells. These results identify the sequence between amino acids 40-82 as an important region within TGF-beta that functions to specify a TGF-beta 1- or TGF-beta 2-like activity.

Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts.

In this report, we have investigated the secretion and synthesis of transforming growth factor-beta 1 (TGF-beta 1) by human dermal fibroblast cultures in response to hypoxia (2% oxygen), and have compared it to standard oxygen culture conditions (15% oxygen at the cell surface). Sandwich enzyme-linked immunosorbent assay (SELISA) showed a selective and progressive increase in secretion of the TGF-beta 1 isoform in response to hypoxia, up to ninefold after cultures were exposed to low oxygen for 72 h; TGF-beta 2 peptide levels were not increased. We then investigated the transcriptional regulation of the TGF-beta 1 gene in response to low and standard oxygen tensions. In the first 24-48 h, TGF-beta 1 mRNA levels decreased steadily in both oxygen environments. This mRNA decline continued for up to 72 h in standard oxygen but not in cultures exposed to low oxygen tension. At 72 h, steady-state TGF-beta 1 mRNA levels were 8 times greater in low compared to standard oxygen, and this increase was reversible upon re-exposure of fibroblast cultures to standard oxygen tension for 24 h. Elevated TGF-beta 1 m-RNA levels in both low and standard oxygen declined steadily and with the same half-life after the addition of actinomycin D, suggesting that hypoxia increased TGF-beta 1 transcription rather than mRNA stability. We conclude that low oxygen tension upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts, and leads to increased secretion of this peptide.

A second messenger RNA species of transforming growth factor beta 1 in infarcted rat heart.

Transforming growth factor-beta 1 (TGF-beta 1) is encoded predominantly by a 2.4-kb mRNA in most tissues. However, an additional transcript of 1.9 kb can be detected in rat heart after experimental myocardial infarction caused by ligation of the left coronary artery. This transcript level is significantly higher in infarcted heart tissue than in normal heart tissue, suggesting an important role for this mRNA species in response to injury. Structural characterization of the 1.9-kb mRNA showed that it included the entire coding sequence present in the 2.4-kb TGF-beta 1 mRNA, but also contained an additional nonhomologous 3'-untranslated region (UTR). The junction between the shared and unique 3' sequence in the 1.9-kb mRNA occurred only two nucleotides before the proposed polyadenylation site of the rat TGF-beta 1 2.4-kb mRNA. The unique 3'-UTR and the deduced shortened 5'-UTR in the novel 1.9-kb TGF-beta 1 mRNA suggest different transcriptional and translational regulatory mechanisms under conditions of tissue injury.

Chemical synthesis and cloning of secretin gene.

A DNA duplex coding for the 27 amino acids of secretin has been synthesized and cloned. In designing the sequence of the gene, computer analysis has been applied. The following factors have been considered: selection of codon usage in favour of expression in yeast; design of various sites useful in gene cloning, gene modification and expressed product purification; avoiding the repeat sequences which may interfere in the ligation of the synthetic fragments. The synthesis involved preparation of 12 oligodeoxyribonucleotides (12-mer to 24-mer in length) by phosphate triester and phosphite triester method, purification by polyacrylamide gel electrophoresis (PAGE). A new plasmid pWS1 was constructed by insertion of the enzymatic ligated gene fragment into plasmid pWR13.

The complete nucleotide sequence of the cloned DNA of hepatitis B virus subtype adr in pADR-1.

The complete nucleotide sequence of the cloned hepatitis B virus DNA subtype adr in pADR-1 was determined by Maxam and Gilbert's method. It is 3215 base pairs in size, which is 27 bp longer than the sequence of the adr pHBr330, as reported by Ono et al. The nucleotide difference between pADR-1 and adr pHBr330 is about 2% while those between pADR-1 and adw as well as ayw are 9.3% and 9.7% respectively. In this paper, the heterogeneity and homogeneity of the S gene, the C gene and the other coding regions in pADR-1 and in the other subtypes are compared and discussed.