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D W Rowe

Publications and source records attributed to D W Rowe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability.

AIM: To analyze the influence of the premature termination codon on mRNA transport and stability METHODS: Chondrocyte mRNA was isolated from homozygous and heterozygous nanomelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggrecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1-(-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase II. Visualization of the aggrecan alleles was performed by in situ hybridization. RESULTS: The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-PCR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or collagen type II. A restriction site induced by premature termination codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for both transcripts within the heterozygous nucleus. In situ hybridization showed no abnormal mRNA accumulation. CONCLUSION: This is the first evidence suggesting that the transcript of the mRNA with the premature termination codon within an exon does exit the nucleus.

Aggrecans↗
PubMed1998

1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

Animals↗
PubMed1998

Receptor tyrosine kinase expression in human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (beta), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo.

Bone Marrow Cells↗
PubMed1998

Interleukin-1 represses COLIA1 promoter activity in calvarial bones of transgenic ColCAT mice in vitro and in vivo.

Interleukin-1 (IL-1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast-like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL-1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter-chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5' flanking sequence and 115 bp of 5' untranslated region fused to the CAT reporter. In 48-h calvarial organ cultures, IL-1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose-related manner, with a maximal inhibition of 40-65%. This repression was retained in 5' deletion constructs truncated to-1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL-1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL-1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA promoter regulation by cytokines and other factors.

Animals↗
PubMed1997

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene.

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Animals↗
PubMed1997

Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts.

BACKGROUND: Marrow stromal fibroblasts (MSFs) are known to contain bone precursor cells. However, the osteogenic potential of human MSFs has been poorly characterized. The aim of this study was to compare the osteogenic capacity of mouse and human MSFs after implantation in vivo. METHODS: After in vitro expansion, MSFs were loaded into a number of different vehicles and transplanted subcutaneously into immunodeficient mice. RESULTS: Mouse MSFs transplanted within gelatin, polyvinyl sponges, and collagen matrices all formed a capsule of cortical-like bone surrounding a cavity with active hematopoiesis. In transplants of MSFs from transgenic mice harboring type I procollagen-chloramphenicol acetyltransferase constructs, chloramphenicol acetyltransferase activity was maintained for up to 14 weeks, indicating prolonged bone formation by transplanted MSFs. New bone formation by human MSFs was more dependent on both the in vitro expansion conditions and transplantation vehicles. Within gelatin, woven bone was observed sporadically and only after culture in the presence of dexamethasone and L-ascorbic acid phosphate magnesium salt n-hydrate. Consistent bone formation by human MSFs was achieved only within vehicles containing hydroxyapatite/tricalcium phosphate ceramics (HA/TCP) in the form of blocks, powder, and HA/TCP powder-type I bovine fibrillar collagen strips, and bone was maintained for at least 19 weeks. Cells of the new bone were positive for human osteonectin showing their donor origin. HA/TCP powder, the HA/TCP powder-type I bovine fibrillar collagen strips, and HA/TCP powder held together with fibrin were easier to load and supported more extensive osteogenesis than HA/TCP blocks and thus may be more applicable for therapeutic use. CONCLUSIONS: In this article, we describe the differences in the requirements for mouse and human MSFs to form bone, and report the development of a methodology for the consistent in vivo generation of extensive bone from human MSFs.

Animals↗
PubMed1996

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

Animals↗
PubMed1996

Nuclear retention of COL1A1 messenger RNA identifies null alleles causing mild osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone fragility. Most cases of severe OI result from mutations in the coding region of the COL1A1 or COL1A2 genes yielding an abnormal collagen alpha chain. In contrast, many patients with mild OI show evidence of a null allele due to a premature stop mutation in the mutant RNA transcript. We have previously described a null allele arising from a splice donor mutation where the transcript containing the included intron was sequestered in the nucleus. Here we demonstrate that transcripts from null alleles arising from premature stop mutations are also present in the nucleus and absent in the cytoplasm. Using reverse transcriptase-PCR and single-strand conformational polymorphism of COL1A1 mRNA from patients with mild OI, we describe three patients with distinct null producing mutations identified from the mutant transcript within the nuclear compartment. A fourth patient with a Gly--->Arg expressed point mutation exhibits the mutant transcript in both compartments. Defining the distribution of allelic variants of COL1A1 mRNA in the nuclear and cytoplasmic compartments gives further insight into cell biology of OI and provides a strategy for investigating potential causes of a null allele.

Adult↗
PubMed1996

Infusions of lidocaine into the amygdala, but not the preoptic area, block pseudopregnancy in the rat.

In the rat, vaginocervical stimulation (VCS) received during mating is required for the subsequent expression of 10-12 days of twice-daily prolactin surges that are necessary for pregnancy or pseudopregnancy (PSP). This temporal separation of sensory stimulus and neuroendocrine response suggests that a mnemonic of the vaginocervical stimulation is created in the brain that triggers and sustains the daily prolactin surges. We investigated the possible involvement of the medial preoptic area (mPOA) and the medial amygdala (mAMYG) as potential neural sites involved in the processing of this neuroendocrine arc. Cycling female rats were bilaterally implanted with intracerebral cannulae in either the mPOA or mAMYG. On proestrus, females were manually palpated to confirm sexual receptivity and then received bilateral infusions of either the local anesthetic lidocaine, the Ca(++) channel blocker, verapamil, or phosphate-buffered saline (PBS) into either brain site before or both before and after receipt of 15 intromissions from an experienced male. Unmated control females received comparable infusions of lidocaine or verapamil, and were placed in the empty test arena for 10 min. Infusions consisted of either a single bilateral infusion 15 min before mating (Expt. 1), bilateral infusions both 15 min before and after mating (Expt. 2) or eight bilateral infusions separated by 30 min intervals spanning a period beginning 45 min before and ending 2 h 45 min after mating (Expt. 3). None of the lidocaine infusions into the mPOA prevented the establishment of PSP, and neither verapamil infusions into the mAMYG nor the shorter-term neural block (i.e. single or double lidocaine infusions) of the mAMYG prevented mating-induced PSP. However, the longer-term neural block (i.e. multiple lidocaine infusions) of the mAMYG significantly reduced the incidence of PSP. These data support previous findings that the mAMYG receives sensory input from VCS, and suggest that the mAMYG is a site at which a mnemonic of VCS is established.

Amygdala↗
PubMed1995

Differential regulation of COL2A1 expression in developing and mature chondrocytes.

To investigate the regulation of type II collagen gene expression in cells undergoing chondrogenic differentiation, we have employed a 5-kbp genomic fragment of the human type II collagen gene which contains 1.8kbp of upstream sequences, the transcription start site, the first exon and 3 kbp of intronic sequences, fused to either lac Z or chloramphenicol acetyl transferase-reporter gene. Transient expression studies revealed a parallel increase in transgene activity and endogenous type II collagen mRNA levels during the onset of the cartilage differentiation of limb mesenchymal cells in high-density micromass cultures. At later periods in culture, however, the transgene activity declines, although steady-state levels of type II collagen mRNA are reported to continue to increase (Kosher et al.: J. Cell. Biol. 102: 1151-1156, 1986; Kravis and Upholt. Dev. Biol. 108: 164-172, 1985). In addition, the activity of the transgene is seven-fold higher at the onset of chondrogenic differentiation in micromass cultures that in well differentiated sternal chondrocytes, although similar levels of type II collagen transcripts are found in these cells. Furthermore, deletions of intronic segments resulted in greater drop in activity of the constructs in differentiating chondrocytes in micromass cultures than in mature sternal chondrocytes. The expression of the construct in transgenic mice is higher at the onset of chondrogenic differentiation and in newly differentiated chondrocytes than in more mature differentiated chondrocytes. Based on these observations, it appears that the mechanisms involved in the regulation of the type II collagen gene at the onset of chondrocyte differentiation are different from those resulting in the maintenance of its expression in fully differentiated chondrocytes.

Animals↗
PubMed1994

Bleomycin stimulates pro-alpha 1 (I) collagen promoter through transforming growth factor beta response element by intracellular and extracellular signaling.

The role of transforming growth factor beta as a mediator of the fibrogenic effect of bleomycin in lung has been investigated at the transcriptional level. Several constructs containing the rat pro-alpha 1 (I) collagen promoter fused to the chloramphenicol acetyltransferase gene were transfected into rat lung fibroblasts. Both bleomycin and transforming growth factor beta 1 increased promoter activity in fibroblasts transfected with constructs containing the transforming growth factor beta response element. Fibroblasts transfected with a deletion construct that lacks this response element did not respond to either bleomycin or transforming growth factor beta 1. Anti-transforming growth factor beta 1-neutralizing antibodies did not block the increase in promoter activity induced by bleomycin, suggesting intracellular signaling. Mutation of the transforming growth factor beta response element greatly reduced the bleomycin effect, which also infers intracellular signaling. In addition, plasmin added to the media greatly enhanced bleomycin stimulation of promoter activity demonstrating that transforming growth factor beta mediates the bleomycin effect through extracellular signaling.

Animals↗
PubMed1994

Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice.

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.

Animals↗
PubMed1994

Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.

3T3 Cells↗
PubMed1994

Molecular basis of nanomelia, a heritable chondrodystrophy of chicken.

Nanomelia is a recessively inherited connective tissue disorder of chicken affecting cartilage development. Other investigators have demonstrated that it involves low aggrecan production and diminished aggrecan mRNA levels. Based on genetic linkage studies showing a high likelihood that the mutation responsible for the nanomelic phenotype lay within the aggrecan gene, a series of experiments was performed to define the molecular basis of the trait. Aggrecan mRNA was present in the nucleus of the nanomelic chondrocyte but greatly reduced in the cytoplasmic compartment, a finding suggestive of a premature stop codon within the aggrecan transcript. Since no defect in mRNA splicing could be demonstrated by ribonucleasease protection studies, direct DNA sequencing was initiated by polymerase chain reaction of the mRNA and of genomic DNA. A stop codon was demonstrated at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon. The mutation creates a unique BasBI restriction site which readily distinguishes the mutant and wild-type alleles.

Aggrecans↗
PubMed1994

Measurement of interleukin-1 stimulated constitutive prostaglandin G/H synthase (cyclooxygenase) mRNA levels in osteoblastic MC3T3-E1 cells using competitive reverse transcriptase polymerase chain reaction.

To assess regulation of constitutive prostaglandin G/H synthase (PGHS-1) by interleukin-1 (IL-1) in osteoblastic MC3T3-E1 cells, we compared analysis by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with Northern blot analysis. Using RT-PCR, IL-1 increased PGHS-1 mRNA levels by 1.84 +/- 0.10 or 2.07 +/- 0.17, depending on the method of calculation. Using Northern blot analysis, the effect of IL-1 on PGHS-1 mRNA levels was more variable, and the variability was increased by normalization of PGHS-1 mRNA levels to the housekeeping genes, beta-actin and glyceraldehyde phosphate dehydrogenase (GAPDH), because their mRNA levels were also regulated by IL-1. We conclude that competitive RT-PCR is a reproducible and accurate method for studying small changes in mRNA levels.

Animals↗