Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.     

Differential expression of TGF beta isoforms in murine palatogenesis

We have studied the expression of genes encoding transforming growth factors (TGFs) beta 1, beta 2 and beta 3 during development of the secondary palate in the mouse from 11.5 to 15.5 days postcoitum using in situ hybridisation. The RNA detected at the earliest developmental stage is TGF beta 3, which is localised in the epithelial component of the vertical palatal shelf. This expression continues in the horizontal palatal shelf, predominantly in the medial edge epithelium, and is lost as the epithelial seam disrupts, soon after palatal shelf fusion. TGF beta 1 RNA is expressed with the same epithelial pattern as TGF beta 3, but is not detectable until the horizontal palatal shelf stage. TGF beta 2 RNA is localised to the palatal mesenchyme underlying the medial edge epithelia in the horizontal shelves and in the early postfusion palate. The temporal and spatial distribution of TGF beta 1, beta 2 and beta 3 RNAs in the developing palate, together with a knowledge of in vitro TGF beta biological activities, suggests an important role for TGF beta isoforms in this developmental process.     

Transforming growth factor-beta: multifunctional regulator of differentiation and development

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Stress fiber growth and remodeling determines cellular morphomechanics under uniaxial cyclic stretch

Biomechanics and Modeling in Mechanobiology - Stress fibers in the cytoskeleton are essential in maintaining cellular shape and influence cellular adhesion and migration. Cyclic uniaxial stretching...     

Synthesis and gene transfection efficacies of PEI-cholesterol-based lipopolymers

Nine lipopolymers based on low molecular weight polyethyleneimines (PEI) and cholesterol via an ether linkage between the polymer amine and the cholesterol backbone have been synthesized. Different percentage of cholesterol moieties have been grafted on three types of PEI of molecular weights 800, 1200, and 2000. These lipopolymers were studied for gene transfection activities in HeLa cells. All the lipopolymers were first optimized for enhanced transfection efficacies as coliposomes with DOPE. All lipopolymers are better transfecting agents and highly serum compatible than commercially available PEI-25KDa. Transfection efficacies and serum compatibility of lipopolymers were found to be dependent upon the MW of PEI used for lipopolymer synthesis and percentage of cholesterol grafting on lipopolymers. Cell viability assay showed that PEI-25KDa is highly toxic as compared to all the lipopolymers. Lipopolyplexes were characterized by transmission electron microscopy, which showed the presence of spherical aggregates.     

Structure-activity investigation on the gene transfection properties of cardiolipin mimicking gemini lipid analogues

A structure-activity relationship has been explored on the gene transfection efficiencies of cardiolipin mimicking gemini lipid analogues upon variation of length and hydrophilicity of the spacer between the cationic ammonium headgroups and lipid hydrocarbon chain lengths. All the gemini lipids were found to be highly superior in gene transfer abilities as compared to their monomeric lipid and a related commercially available formulation. Pseudoglyceryl gemini lipids bearing an oxyethylene (-CH2-(CH2-O-CH2)m-CH2-) spacer were found to be superior gene transfecting agents as compared to those bearing polymethylene (-CH2)m-) spacers. The major characteristic feature of the present set of gemini lipids is their serum compatibility, which is most often the major hurdle in liposome-mediated gene delivery.     

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.