Biomedical subjects

D W Rowe

Publications and source records attributed to D W Rowe.

At least 55 records · Page 3Linked to original sources

PubMed1993

c-Fos proto-oncogene activity induced by mating in the preoptic area, hypothalamus and amygdala in the female rat: role of afferent input via the pelvic nerve.

In order to identify brain areas which receive afferent genitosensory input important for mating-induced prolactin release, we compared numbers of Fos-immunoreactive (Fos-IR) cells in brains of intact estrous females 1 h after differential mating stimulation. Numbers of Fos-IR cells were approximately 3-fold higher in the preoptic area (POA), medial amygdala (mAMYG) and bed nucleus of the stria terminalis (BNST) when females received intromissions (I) from males than when they received mounts-without-intromission (M) or were taken directly from their home cage. In the ventrolateral portion of the ventromedial nucleus (VL-VMN), the paraventricular nucleus (PVN) of the hypothalamus and the midbrain central tegmental field (CTF) numbers of Fos-IR cells were significantly higher than home cage levels in groups of females exposed to males regardless of type of mating stimulation received. Bilateral transection of the pelvic nerve eliminated the increases in Fos-IR in POA and mAMYG which occurred in sham-transected females in these areas after intromissions from males. These data demonstrate that afferent input via the pelvic nerve activates cell groups within the POA, mAMYG and BNST and suggests that these areas may be involved in initiation of mating-induced prolactin surges.

Afferent Pathways↗

PubMed1993

Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue associated with fractures, osteopenia, and short stature. OI results from mutations affecting the pro alpha 1 or pro alpha 2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human OI. The phenotype of homozygous oim mice includes skeletal fractures, limb deformities, generalized osteopenia, and small body size. Their femurs are smaller and demonstrate marked cortical thinning and fewer medullary trabeculae than those of wild-type mice. Breeding studies show the mutation is inherited in most crosses as a single recessive gene on chromosome 6, near the murine Cola-2 gene. Biochemical analysis of skin and bone, as well as isolated dermal fibroblast cultures, demonstrate that alpha 1(I) homotrimeric collagen accumulates in these tissues and is secreted by fibroblasts. Short labeling studies in fibroblasts demonstrate an absence of pro alpha 2(I) collagen chains. Nucleotide sequencing of the cDNA encoding the COOH-propeptide reveals a G deletion at pro alpha 2(I) nucleotide 3983; this results in an alteration of the sequence of the last 48 amino acids. The oim mouse will facilitate the study of type I collagen-related skeletal disease.

Amino Acid Sequence↗

PubMed1993

Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells.

To directly compare the patterns of collagen promoter expression in cells and tissues, the activity of COL1A1 fusion genes in calvariae of neonatal transgenic mice and in primary bone cell cultures derived by sequential digestion of transgenic calvariae was measured. ColCAT3.6 contains 3.6 kb (positions -3521 to +115) of the rat COL1A1 gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. ColCAT2.3 and ColCAT1.7 are 5' deletion mutants which contain 2,296 and 1,672 bp, respectively, of COL1A1 DNA upstream from the transcription start site. ColCAT3.6 activity was 4- to 6-fold lower in primary bone cell cultures than in intact calvariae, while ColCAT2.3 activity was at least 100-fold lower in primary bone cells than in calvariae. These changes were accompanied by a threefold decrease in collagen synthesis and COL1A1 mRNA levels in primary bone cells compared with collagen synthesis and COL1A1 mRNA levels in freshly isolated calvariae. ColCAT3.6 and ColCAT2.3 activity was maintained in calvariae cultured in the presence or absence of serum for 4 to 7 days. Thus, when bone cells are removed from their normal microenvironment, there is parallel downregulation of collagen synthesis, collagen mRNA levels, and ColCAT3.6 activity, with a much greater decrease in ColCAT2.3. These data suggest that a 624-bp region of the COL1A1 promoter between positions -2296 and -1672 is active in intact and cultured bone but inactive in cultured cells derived from the bone. We suggest that the downregulation of COL1A1 activity in primary bone cells may be due to the loss of cell shape or to alterations in cell-cell and/or cell-matrix interactions that normally occur in intact bone.

Animals↗

PubMed1993

Defective splicing of mRNA from one COL1A1 allele of type I collagen in nondeforming (type I) osteogenesis imperfecta.

Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COL1A1 gene. We studied fibroblasts established from a child with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1 (I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population was confined to the nuclear compartment of the cell, and contained the entire sequence of intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A transition was also identified in the genomic DNA. The retained intron contained an in-frame stop codon and introduced an out-of-frame insertion within the collagen mRNA producing stop codons downstream of the insertion. These changes probably account for the failure of the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript, this mutation did not result in production of a defective collagen pro alpha 1 (I) chain. Instead, the mild nature of the disease in this case reflects failure to process the defective mRNA and thus the absence of a protein product from the mutant allele.

Adolescent↗

PubMed1993

Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice.

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.

Animals↗

PubMed1993

[Primary management of polytrauma. Comparison of a German and American air rescue unit].

Hospital-based helicopter services from German and American university-affiliated trauma centers were reviewed. All multitrauma patients transported via helicopter from the scene of the accident to the trauma center during a 1-year period were included. The patients were comparable regarding mechanism of injury, age, flight times, mean ISS, ISS distribution, and number of severe injuries per body region (patients with AIS > 3 for head, thorax and abdomen). Overall mortality for the German system was 21/221 (9.5%) and 21/186 (11.3%) for the American system (not significant). Survivor-based TRISS analysis yielded Z-statistics of +2.459 for the German, and +1.049 for the American system. There were 9 unexpected survivors (Ps < 0.5) in the German, 6 in the American system. There was a significant higher (P < 0.01) number of early deaths (< 6 h) in the American population (12, ISS 56) than in the German (4, ISS 64). Analysis of the prehospital data demonstrated significant differences in the mean volume of IV fluids infused: 1800 cc German, 825 cc American (P < 0.05); rate of intubation: 82/221 (37.1%) German, 24/186 (13.4%) American (P < 0.001); and thoracic decompressions: 20/221 (9.1%) German, 1/186 (0.5%) American (P < 0.001). Pre-hospital care in the German system is directed on-scene by a trauma surgeon member of the flight crew, compared to a nurse/paramedic team with remote medical control in the American system. Compared to an American trauma system, the German system demonstrates improved overall outcome as measured by survivor-based TRISS Z-statistics. More favorable German Z-statistics are in part related to fewer early deaths.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV.

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.

Alkaline Phosphatase↗

PubMed1992

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

3T3 Cells↗

PubMed1992

On-scene helicopter transport of patients with multiple injuries--comparison of a German and an American system.

Hospital-based helicopter services from a German (GER) and an American (AMR) university-affiliated trauma center were reviewed. All patients with multiple injuries transported via helicopter from the scene to the trauma centers during a 1-year period were included. The patients were comparable regarding mechanism of injury, age, flight times, mean ISS, ISS distribution, and number of severe injuries per body region (patients with AIS score > 3 for head, thorax, and abdomen). Overall mortality was 21 of 221 (9.5%) for GER and 21 of 186 (11.3%) for AMR (NS). Survivor-based TRISS analysis yielded Z statistics of +2.459 for GER (p < 0.025) and +1.049 for AMR (NS). M statistics were 0.89 for GER, 0.874 for AMR; the W statistic +1.35 for GER. There were nine unexpected survivors (Ps < 0.50) for GER and six for AMR. There was a significantly higher (p < 0.01) number of early deaths (< 6 hours) in AMR (12; ISS = 56) than in GER (four; ISS = 64). Analysis of the prehospital data demonstrated significant differences in the mean volume of IV fluids infused: 1800 mL, GER; 825 mL, AMR (p < 0.05); rate of intubation: 82 of 221 (37.1%) GER; 24 of 186 (13.4%) AMR (p < 0.001); and thoracic decompressions: 20 of 221 (9.1%) GER; 1 of 186 (0.5%) AMR (p < 0.001). Prehospital care in the GER system is directed on scene by a trauma surgeon member of the flight crew compared with a nurse/paramedic team with remote medical control in the AMR system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

An osteopenic nonfracture syndrome with features of mild osteogenesis imperfecta associated with the substitution of a cysteine for glycine at triple helix position 43 in the pro alpha 1(I) chain of type I collagen.

Mutations affecting the pro alpha 1(I) or pro alpha 2(I) collagen genes have been identified in each of the major clinical types of osteogenesis imperfecta. This study reports the presence of a heritable connective tissue disorder in a family with an osteopenic syndrome which has features of mild osteogenesis imperfecta but was considered idiopathic osteoporosis in the proband. At age 38, while still premenopausal, she was found to have osteopenia, short stature, hypermobile joints, mild hyperelastic skin, mild scoliosis, and blue sclerae. There was no history of vertebral or appendicular fracture. Hip and vertebral bone mineral density measurements were consistent with marked fracture risk. Delayed reduction SDS-PAGE of pepsin-digested collagens from dermal fibroblast cultures demonstrated an anomalous band migrating between alpha 1(I) and alpha 1(III). This band merged with the normal alpha-chains upon prereduction, indicating an unexpected cysteine residue. Cyanogen bromide peptide mapping suggested that the mutation was in the smaller NH2-terminal peptides. cDNA was reverse transcribed from mRNA and amplified by the polymerase chain reaction. A basepair mismatch between proband and control alpha 1(I) cDNA hybrids was detected by chemical cleavage with hydroxylamine:piperidine. The cysteine substitution was thus localized to alpha 1(I) exon 9 within the cyanogen bromide 4 peptide. Nucleotide sequence analysis localized a G----T point mutation in the first position of helical codon 43, replacing the expected glycine (GGT) residue with a cysteine (TGT). The prevalence of similar NH2-terminal mutations in subjects with this phenotype which clinically overlaps idiopathic osteoporosis remains to be determined.

Adolescent↗

PubMed1991

Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene.

We have located a cis-acting element (alpha 1-TAE) within the promoter sequences of the rat collagen alpha 1(I) gene (COL1A1) 1600 bases upstream of the transcription start site which mediates transcriptional activation by transforming growth factor beta (TGF-beta). The functional significance of this region was established by (1) deletion analysis of the alpha 1(I) promoter cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and (2) by co-transfection of promoter constructs with double-stranded oligonucleotides. DNA-mobility-shift assays with radiolabelled alpha 1-TAE demonstrated increased nuclear binding activity after TGF-beta stimulation. Oligonucleotides encoding the alpha 1-TAE, additional upstream regions within the alpha 1(I) promoter, as well as consensus nuclear-factor-1 (NF-1) sequences, competed with the alpha 1-TAE sequence. The two collagen type I genes are stimulated by TGF-beta through different regions of their promoters.

Animals↗

PubMed1991

Cell adhesion regulates pro-alpha 1(I) collagen mRNA stability and transcription in mouse fibroblasts.

Adhesive interactions are important modulators of cellular phenotype. Previously, we demonstrated that quiescent, suspension-arrested cells are not equivalent to density-arrested cells in their patterns of gene expression (Dhawan, J., and Farmer, S.R. (1990) J. Biol. Chem. 265, 9015-9021). In particular, pro-alpha 1(I) collagen expression depended strongly on the extent of cell adhesion. In this paper, we demonstrate that the adhesion-induced rise in collagen gene expression is due to regulation at multiple levels. Steady state levels of pro-alpha 1(I) collagen mRNA increased up to 10-fold by 6 h after replating suspended cells, and this rise is blocked by inhibition of protein synthesis. Transcription of the pro-alpha 1(I) collagen gene was measured by run-on assay as well as by activation of a rat alpha 1(I) promoter-chloramphenicol acetyltransferase reporter gene construct. Both assays reveal a 5-fold depression of pro-alpha 1(I) collagen gene transcription in suspended cells. Reattachment of suspended cells resulted in the activation of alpha 1(I) gene transcription by 2-h postreplating, reaching a 3-5-fold level of induction by 18 h. The pro-alpha 1(I) collagen mRNA was substantially more labile in suspended cells than in adherent cells (t1/2 values of approximately 2 h in nonadherent cells and greater than 8 h in exponentially growing or density-arrested cells). Furthermore, reattachment of suspended cells for 18 h resulted in a stabilization of collagen mRNA. We conclude that cell adhesion regulates pro-alpha 1(I) collagen gene expression selectively and at transcriptional and posttranscriptional sites.

Animals↗

PubMed1990

Cloning and analysis of the 5' region of the rat bone/liver/kidney/placenta alkaline phosphatase gene. A dual-function promoter.

The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2 kb of 5' flanking region, its first exon (84bp), and over 7 kb of the first intron. The promoter of the rat BLKP ALP displays features of a "housekeeping" gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions-134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85 kb promoter region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer. The homologous human bone/liver/kidney (BLK) ALP promoter, which demonstrates a similar combination of tissue-specific and housekeeping characteristics, shares close similarity (184 bp of 79% similarity excluding gaps) with the rat BLKP ALP promoter. The human placental ALP is encoded by a separate gene and its promoter, on the other hand lacks significant similarity to the rat BLKP ALP promoter despite their common expression in the placenta. This lack of similarity appears to reflect the close evolutionary relationship of the human placental ALP gene to the intestinal ALP gene. Significant sequence similarity was found between the rat and human BLK/BLKP ALP promoters and the human and mouse adenine deaminase promoters, and together they may represent a class of dual-function promoters, allowing both constitutive low-level, and tissue-specific higher levels of expression. A pentanucleotide with the consensus sequence 5'-GGCTC-3' is present in these promoters and in the promoters for the human fibronectin and the human alpha 1(II) procollagen genes in the region of maximal similarity with the rat BLKP ALP promoter, and in the vicinity of the Sp1-binding sites.

Alkaline Phosphatase↗

PubMed1989

Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D.

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.

Animals↗

PubMed1989

Expression of chloramphenicol acetyltransferase (CAT) from rodent type I collagen promoters in transfected osteosarcoma cells in vivo.

We examined the osteoblastic phenotype of permanently transfected ROS 17/2.8 cells in culture and in vivo, in order to evaluate their relevance for studies of the regulation of gene expression and gene function in osteoblastic cells. Recent reports indicate that the progeny transfected cells may substantially vary and differ from the parental cell line in their phenotype, particularly in their tumorigenicity. ROS 17/2.8 cells were transfected with genetic constructs expressing the CAT gene from either the rat alpha 1 (I) or the mouse alpha 2 (I) collagen promoters. Forty-four clonal cell lines display a range of CAT expression from the transfected collagen promoters in culture. Four of these cell lines were further characterized. Alkaline phosphatase activity in these four cell lines is higher than in fibroblastic cells. These four cell lines are tumorigenic in immunocompatible ACI rats and form calcified tumors similar to those formed by ROS 17/2.8. CAT expression could be demonstrated in tumor extracts of two of the four cell lines, which also expressed higher CAT levels in culture. We conclude that permanently transfected ROS 17/2.8 derived cell lines maintain their tumorigenicity and their osteoblastic-like phenotype, and thus may provide a useful system for studies of gene function and regulation in osteoblast-like cells and bone-like tissue in vivo.

Animals↗

PubMed1989

1,25-Dihydroxyvitamin D3 inhibits transcription of type I collagen genes in the rat osteosarcoma cell line ROS 17/2.8.

Our previous studies have demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3)] reduces type I collagen synthesis and steady state levels of procollagen mRNA in cultured fetal rat calvaria and rat osteosarcoma cells. To determine whether 1,25-(OH)2D3 regulates transcription of type I collagen genes, transcription rates were measured directly in nuclei isolated from ROS 17/2.8 cells using a nuclear run-off assay. Transcription was allowed to proceed in the presence of [32P]UTP for 20 min, at which time incorporation of radiolabeled UTP into trichloroacetic acid-precipitable material was maximal. UTP incorporation was inhibited 90% by 3 micrograms/ml actinomycin-D and 40% by 1 microgram/ml alpha-amanitin. Treatment of ROS 17/2.8 cells with 1,25-(OH)2D3 inhibited procollagen gene transcription in a concentration and time dependent manner. Procollagen transcription was reduced by approximately 50% of the control rate by 10 nM 1,25-(OH)2D3, and this inhibition was maximal after 24 h of 1,25-(OH)2D3 treatment. The inhibition of procollagen transcription was specific for collagen, since total RNA synthesis and beta-actin transcription were not inhibited by 1,25-(OH)2D3. The magnitude of the decrease of procollagen transcription by 1,25-(OH)2D3 was comparable to its inhibition of steady state procollagen mRNA levels, suggesting that transcription is the predominant mechanism by which 1,25-(OH)2D3 regulates collagen gene expression in bone cells.

Animals↗

PubMed1989

Insulin increases the steady state level of alpha-1(I) procollagen mRNA in the osteoblast-rich segment of fetal rat calvaria.

Insulin at 3-100 nM increased the steady state level of alpha-1(I) procollagen mRNA and stimulated collagen synthesis in the osteoblast-rich segment of central bone from 21-day-old fetal rat calvaria. The increases in the level of procollagen mRNA and the rate of collagen synthesis were observed 18 h after the addition of insulin to the cultures. The removal of insulin from calvaria incubated for 24 h with 3 nM insulin caused collagen synthesis and the level of alpha-1(I) procollagen mRNA to return to control values within 5 h. Adding insulin back to calvaria withdrawn from insulin treatment for 3 h did not rescue the decay in collagen synthesis or the level of alpha-1(I) procollagen mRNA. Insulin increased the steady state levels of alpha-1(I) procollagen mRNA in the presence of the RNA synthesis inhibitor actinomycin-D. Our data suggest that in fetal rat bone, one mechanism by which insulin increases the steady state level of alpha-1(I) procollagen mRNA may be by altering its stability.

Animals↗

PubMed1988

Malignant osteoporosis and defective immunoregulation.

The linkage between immune cells and the osteoclast has become partially understood in the laboratory, but the full spectrum of clinical disorders of this relationship remain to be elucidated. We report a 29-month-old girl with recurrent infections and multiple fractures. Immune evaluation showed normal quantitative serum immunoglobulins but absent antibodies to the respiratory viruses and tetanus toxoid and decreased in vitro polyclonal-induced immunoglobulin production. Further analysis in vitro with separated lymphocyte populations showed normal B cell function but markedly increased suppressor T cell activity. The bone evaluation showed diffuse osteopenia on x-ray. Serum calcium, phosphorus, PTH, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were normal for age. Urinary calcium excretion (24 h) was, however, two times normal. An iliac crest biopsy confirmed the presence of extreme osteopenia with normal mineralization and numerous small atypical osteoclasts resorbing the bone. No circulating plasma resorptive activity was demonstrated. Calcitonin therapy markedly diminished the patient's hypercalciuria. We speculate that this patient's increased bone resorption, decreased bone formation, and suppressor activity may be linked by a common pathway involving the abnormal function of immune cells. Since no similar constellation of findings has been previously reported, this case may represent a new congenital disorder: severe osteopenia associated with increased osteoclast activity in association with a defect in T cell immunoregulation.

Biopsy↗