Mesenchymal stem cells used for rabbit tendon repair can form ectopic bone and express alkaline phosphatase activity in constructs.

Mesenchymal stem cells (MSCs) have been used to repair connective tissue defects in several animal models. Compared to "natural healing" controls (no added cells), MSC-collagen gel constructs in rabbit tendon defects significantly improve repair biomechanics. However, ectopic bone forms in 28% of MSC-treated rabbit tendons. To understand the source of bone formation, three studies were performed. In the first study, the hypothesis was tested that MSCs delivered during surgery contribute to bone formation in the in vivo repair site. Adjacent histological sections in the MSC-treated repair tissue were examined for pre-labeled MSCs and for cells showing positive alkaline phosphatase (ALP) activity. Both cells were observed in serial sections in regions of ectopic bone. Contralateral "natural healing" tendons lacked both markers. In the other two studies, the effects of osteogenic supplements and construct geometry (monolayer vs. 3-D) on ALP activity were studied to test three hypotheses: that rabbit MSCs increase ALP activity over time in monolayer culture conditions; that adding osteogenic inducing supplements to the culture medium increases cellular protein in monolayer culture; and that rabbit MSCs increase ALP activity both in monolayer and in 3-D constructs, with and without media supplements. Culture in monolayer under similar conditions to in vivo (as in the first study) did not increase ALP at 2 or 4 weeks. Medium designed to increase osteogenic activity significantly increased cell numbers (cellular protein increased by 260%) but did not affect ALP activity either in monolayer or 3-D constructs (p>0.12). However, MSCs in 3-D constructs exhibited higher ALP activity than cells in monolayer, both in the presence (p<0.045) and absence of supplement (p<0.005). These results suggest that in vitro conditions may critically influence cell differentiation and protein expression. Mechanisms responsible for these effects are currently under investigation.

Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta.

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

Alignment and proliferation of MC3T3-E1 osteoblasts in microgrooved silicone substrata subjected to cyclic stretching.

Previous studies have shown that many types of cells align in microgrooves in static cultures. However, whether cells remain aligned and also proliferate in microgrooves under stretching conditions has not been determined. We grew MC3T3-E1 osteoblasts in deformable silicone dishes containing microgrooves oriented in the stretch direction. We found that with or without 4% stretching, cells aligned in microgrooves of all sizes, with the groove and ridge widths ranged from 1 to 6microm, but the same groove depth of about 1.6microm. In addition, actin cytoskeleton and nuclei became highly aligned in the microgrooves with and without 4% cyclic stretching. To further examine whether MC3T3-E1 osteoblasts proliferate in microgrooves with cyclic stretching, we grew the cells in six-well silicone dishes containing microgrooves in three wells and smooth surfaces in other three wells. After 4% cyclic stretching for 3, 4, and 7 days, we found that cell numbers in the microgrooves were not significantly different (p>0.05) from those on the smooth surface (p>0.05). Taken together, these results show that MC3T3-E1 osteoblasts can align and proliferate in microgrooves with 4% cyclic stretching. We suggest that the silicone microgrooves can be a useful tool to study the phenotype of MC3T3-E1 osteoblasts under controlled substrate strains. The silicone microgrooves can also be useful for delivering defined substrate strains to other adherent cells in cultures.

COL5A1 haploinsufficiency is a common molecular mechanism underlying the classical form of EDS.

We have identified haploinsufficiency of the COL5A1 gene that encodes the proalpha1(V) chain of type V collagen in the classical form of the Ehlers-Danlos syndrome (EDS), a heritable connective-tissue disorder that severely alters the collagen-fibrillar structure of the dermis, joints, eyes, and blood vessels. Eight of 28 probands with classical EDS who were heterozygous for expressed polymorphisms in COL5A1 showed complete or nearly complete loss of expression of one COL5A1 allele. Reduced levels of proalpha1(V) mRNA relative to the levels of another type V collagen mRNA, proalpha2(V), were also observed in the cultured fibroblasts from EDS probands. Products of the two COL5A1 alleles were approximately equal after the addition of cycloheximide to the fibroblast cultures. After harvesting of mRNAs from cycloheximide-treated cultured fibroblasts, heteroduplex analysis of overlapping reverse transcriptase-PCR segments spanning the complete proalpha1(V) cDNA showed anomalies in four of the eight probands that led to identification of causative mutations, and, in the remaining four probands, targeting of CGA-->TGA mutations in genomic DNA revealed a premature stop at codon in one of them. We estimate that approximately one-third of individuals with classical EDS have mutations of COL5A1 that result in haploinsufficiency. These findings indicate that the normal formation of the heterotypic collagen fibrils that contain types I, III, and V collagen requires the expression of both COL5A1 alleles.

Discordant expression of osteoblast markers in MC3T3-E1 cells that synthesize a high turnover matrix.

To examine the autocrine effects that an organizing extracellular matrix has on osteoblast precursors, we created MC3T3-E1 cell lines that stably expressed pro-alpha1(I) collagen chains with a truncated triple helical domain. Cells that had incorporated the pro-alpha1(I) expression plasmid (pMG155) expressed shortened pro-alpha1(I) transcripts at high levels and efficiently secreted the expression gene products into culture media. Those cells lost over 30% of newly deposited collagenous matrix compared with virtually no loss in control cultures, and media from the abnormal cells had qualitative differences in matrix metalloprotinase production. Electron micrographs strongly suggested that type I collagen molecules containing the truncated pro-alpha1(I) chains dramatically interfered with collagen fibrillogenesis in newly forming osteoblast matrix. Abnormal collagen fibrillogenesis was also associated with altered characteristics of cellular differentiation in that abnormal cells displayed a delayed and attenuated increase in alkaline phosphatase activity. Surprisingly, synthesis of osteocalcin was more than 5-fold higher than control cultures. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for up-regulation of alkaline phosphatase activity. However, in the presence of rapid turnover of osteoblast matrix, osteocalcin gene expression may be up-regulated in response to local signals by an unknown mechanism.

Abnormal differentiation in MC3T3-E1 preosteoblasts expressing a dominant-negative type I collagen mutation.

To examine the effects that an organizing extracellular matrix might have on osteoblast precursors, we created MC3T3-E1 cell lines that stably incorporated a plasmid that expressed pro alpha 1 (I) collagen chains having a truncated triple helical domain. Cells that had incorporated the pro alpha 1 (I) expression plasmid (pMG155) efficiently secreted molecules with shortened pro alpha 1 (I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically interferes with normal type I collagen fibril architecture. The turnover of newly deposited collagenous matrix as measured by 3[H]-hydroxyproline release was 29% after a 14 day chase in cells expressing the minigene compared to essentially no turnover in control cultures. MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of cell division followed by an increase in osteoblast characteristics. Cell number was consistently 20-25% higher than control in MC3T3-E1 cultures expressing the truncated pro alpha 1 (I) gene but ALP activity was only 45% of control. Secretion and steady state mRNA levels for osteocalcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for inhibition of cellular proliferation and subsequent upregulation of ALP. However, in the presence of rapid turnover of osteoblast matrix, the gene for osteocalcin may be upregulated in response to local signals.

Isolation of a novel latent transforming growth factor-beta binding protein gene (LTBP-3).

This paper reports the molecular cloning of a novel gene in the mouse that shows structural similarities to the microfibril protein fibrillin and to the latent transforming growth factor-beta (TGF-beta) binding protein (LTBP), a component of the latent TGF-beta complex. The gene was initially isolated during a low stringency polymerase chain reaction screen of a NIH 3T3 cell cDNA library using primers that amplify a human fibrillin-1 epidermal growth factor-like repeat. Three lines of evidence suggest that the mouse gene is a third member of the LTBP gene family, which we designate LTBP-3. First, the deduced polypeptide, which consists of 15 epidermal growth factor-like repeats, 3 TGF binding protein repeats, and 2 proline- and glycine-rich sequences, shows 38.4% identity with LTBP-1 but only 27% identity with fibrillin-1. Second, the gene appears to be co-expressed in developing mouse tissues with TGF-beta. Third, immunoprecipitation studies using mouse preosteoblast MC3T3-E1 cells and a specific anti-peptide polyclonal antiserum reveal that the mouse polypeptide forms a complex with the TGF-beta 1 precursor. Finally, we note that the LTBP-3 gene was recently localized to a distinct genetic locus (Li, X., Yin, W., Perez-Jurado, L., Bonadio, J., and Francke, U. (1995) Mamm. Genome 6, 42-45). Identification of a third binding protein provides further insight into a mechanism by which latent TGF-beta complexes can be targeted to connective tissue matrices and cells.

Characterization of 1,25-dihydroxyvitamin D3 receptors and in vivo targeting of [3H]-1,25(OH)2D3 in the sheep placenta.

We sought to detect the presence of receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in placental tissues of five late gestational pregnant sheep and to quantitate their biochemical properties and abundance. Cytosol prepared from cotyledonary tissue was found to contain two [3H]-1,25(OH)2D3 binding macromolecules that sedimented at 3.2 S and 4.1 S, respectively, on linear (4-20 per cent) hypertonic sucrose gradients. The 4.1 S component cosedimented with serum that had been prelabelled with [3H]-25-hydroxyvitamin D3 (25-OHD3) and was present in cytosols despite extensive washing of the tissue prior to homogenization. Concurrent incubation of the cytosol with [3H]-1,25(OH)2D3 and a tenfold molar excess of radioinert 25-OHD3 resulted in complete resolution of the 3.2 S macromolecule and disappearance of the 4.1 S binding component. The binding of [3H]-1,25(OH)2D3 to the 3.2 S component was completely abolished by coincubation with a 100-fold molar excess of radioinert 1,25(OH)2D3 and was replaced by a well resolved peak in the 4.1 S region. Scatchard analysis of cytosol binding to [3H]-1,25(OH)2D3 in the presence of a tenfold molar excess of radioinert 25OHD3 revealed a single class of non-interacting saturable binding site in the cotyledon and the endometrium of high affinity and low capacity. The mean +/- s.e. of the dissociation constant of the cotyledonary receptor of 0.21 +/- 0.06 nM was not different from that of 0.16 +/- 0.03 nM for the endometrial receptor. However, the abundance of the cotyledonary receptor was fourfold higher than that in the endometrium (110 +/- 20 versus 28 +/- 7 fmol/mg protein). Since it is not possible to completely separate endometrial tissue from cotyledonary tissue, the low abundance of receptor in endometrial cytosols may merely represent contamination of endometrial tissue with cotyledonary tissue. Further analysis of the [3H]-1,25(OH)2D3 occupied receptor in cotyledonary cytosols showed that it bound to DNA cellulose and was eluted with 0.16 M KCl. This in vitro binding of [3H]-1,25(OH)2D3 to DNA was confirmed in vivo by the finding of preferential nuclear targetting of [3H]-1,25(OH)2D3 (56 per cent of total cellular activity), 4 h after fetal intravenous administration of [3H]-1,25(OH)2D3 to five chronically catheterized fetal sheep. Total placental uptake of [3H]-1,25(OH)2D3 at this time amounted to 3.7 +/- 0.9 per cent of the injected dose. Preliminary analysis of ovine placental cytosols revealed a calcium binding protein of similar molecular weight to that found in the ovine intestine and in the intestine and placenta of rodents.(ABSTRACT TRUNCATED AT 400 WORDS)

Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci.

Salmonella typhimurium strains lacking the CorA Mg2+ transport system retain Mg2+ transport and the ability to grow in medium containing a low concentration of Mg2+. Mutagenesis of a corA strain followed by ampicillin selection allowed isolation of a strain that required Mg2+-supplemented media for growth. This strain contained mutations in at least two loci in addition to corA, designated mgtA and mgtB (for magnesium transport). Strains with mutations at all three loci (corA, mgtA, and mgtB) exhibited no detectable Mg2+ uptake and required 10 mM Mg2+ in the medium for growth at the wild-type rate. A wild-type allele at any one of the three loci was sufficient to restore both Mg2+ transport and growth on 50 microM Mg2+. P22 transduction was used to map the mgt loci. The mgtA mutation was located to approximately 98 map units (cotransducible with pyrB), and mgtB mapped at about 80.5 map units (near gltC). A chromosomal library from S. typhimurium was screened for clones that complemented the Mg2+ requirement of a corA mgtA mgtB mutant. The three classes of plasmids obtained could each independently restore Mg2+ transport to this strain and corresponded to the corA, mgtA, and mgtB loci. Whereas the corA locus of S. typhimurium is analogous to the corA locus previously described for Escherichia coli, neither of the mgt loci described in this report appears analogous to the single mgt locus described in E. coli. Our data in this and the accompanying papers (M. D. Snavely, J. B. Florer, C. G. Miller, and M. E. Maguire, J. Bacteriol. 171:4752-4760, 4761-4766, 1989) indicate that the corA, mgtA, and mgtB loci of S. typhimurium represent three distinct systems that transport Mg2+.

Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems.

In Salmonella typhimurium, the corA, mgtA, and mgtB loci are involved in active transport of Mg2+ (S. P. Hmiel, M. D. Snavely, C. G. Miller, and M. E. Maguire, J. Bacteriol. 168:1444-1450, 1988; S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). In this study, the gene products coded for by the corA, mgtA, and mgtB genes were identified by using plasmid expression in Escherichia coli maxicells. Complementation was assessed by introducing plasmids into a Mg2+-dependent corA mgtA mgtB strain and determining the ability of the plasmid to restore growth on medium without a Mg2+ supplement. Complementing plasmids containing corA expressed a 42-kilodalton (kDa) protein. This protein was not expressed by plasmids containing insertions or deletions that eliminated complementation. A plasmid containing mgtA expressed 37- and 91-kDa gene products. Data obtained with subclones and insertions in this plasmid indicated that plasmids expressing only the 91-kDa polypeptide complemented; plasmids that did not express this protein did not complement regardless of whether they expressed the 37-kDa protein. Plasmids carrying mgtB expressed a single protein of 102 kDa whose presence or absence correlated with the ability of the plasmid to complement the Mg2+-dependent triple mutant. Fractionation of labeled maxicells demonstrated that the 42-kDa corA, the 91-kDa mgtA, and the 102-kDa mgtB gene products are all tightly associated with the membrane, a location consistent with involvement in a transport process. These data provide further support the for existence of three distinct systems for Mg2+ transport in S. typhimurium.

Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems.

Three loci in Salmonella typhimurium (corA, mgtA, and mgtB) code for components of distinct Mg2+ transport systems (S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). Strains carrying one wild-type and two mutant alleles of the three loci were constructed to study the kinetics and specificity of ion transport of each system in isolation. The transport systems had different Km and Vmax values for Mg2+ uptake, and each was inhibited by other divalent cations in a distinct rank order of potency: for CorA, Mg2+ greater than Mn2+ greater than Co2+ greater than Ni2+ greater than Ca2+; for MgtA, Zn2+ greater than or equal to Mg2+ greater than Ni2+ approximately Co2+ greater than Ca2+; and for MgtB, Mg2+ approximately Ni2+ approximately Ni2+ greater than Mn2+ much greater than Ca2+. Other differences among the three systems were apparent. The CorA transport system functioned as a Mg2+-Mg2+ exchange system, mediating both efflux and influx of Mg2+. Neither the MgtA nor the MgtB system could mediate Mg2+ efflux. Transport via the MgtB system was very temperature sensitive; Mg2+ was transported at 37 degrees C but not at 20 degrees C. The MgtA and the MgtB transport systems were found to be regulated by the extracellular concentration of Mg2+.