Biomedical subjects

A C Lichtler

Publications and source records attributed to A C Lichtler.

At least 19 recordsLinked to original sources

PubMed2003

Stage specific inhibition of osteoblast lineage differentiation by FGF2 and noggin.

Fibroblast growth factor 2 (FGF2) and noggin are two unrelated ligands of two distinctly different signaling pathways that have a similar inhibitory effect on osteoblast differentiation. Because of their differences, we postulated that they probably acted at a different stage within the osteoprogenitor differentiation pathway. This study was performed on primary murine bone cell cultures under conditions where alkaline phosphatase (AP) and type I collagen expression (Col1a1) were observed by day 7 (preosteoblast stage), followed by bone syaloprotein (BSP) at day 11 (early osteoblast) and osteocalcin (OC) by day 15-18 (mature osteoblast stage). FGF2 completely inhibited expression of AP and the mRNA transcript for Col1a1, while noggin showed only a partial inhibition of these markers of preosteoblast differentiation. However, the markers of differentiated osteoblasts (BSP and OC) were completely inhibited in both the FGF2 and noggin treated cultures, suggesting that noggin acts at later point in the osteoprogenitor differentiation pathway than FGF2. To further verify that the inhibition was occurring at a different stage of osteoblasts development, primary cultures derived from transgenic mice harboring segments of the collagen promoter driving green fluorescent protein (GFP) that activate at different levels of osteoblast differentiation were analyzed. Consistent with the endogenous markers, pOBCol3.6GFP and pOBCOL2.3GFP transgene activity was completely inhibited by continuous addition of FGF2, while noggin showed partial inhibition of pOBCol3.6GFP and complete inhibition of the pOBCol2.3GFP transgene. Upon removal of either agent, endogenous and GFP markers of osteoblast differentiation reappeared although at a different temporal pattern. This work demonstrates that FGF2 and noggin can reversibly modulate osteoblast lineage differentiation at different maturational stages. These agents may be useful to enrich for and maintain a population of osteoprogenitor cells at a defined stage of differentiation.

Animals↗

PubMed2002

Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage.

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

Animals↗

PubMed2001

Use of VSV-G pseudotyped retroviral vectors to target murine osteoprogenitor cells.

Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.

Alkaline Phosphatase↗

PubMed2001

Bone-directed expression of Col1a1 promoter-driven self-inactivating retroviral vector in bone marrow cells and transgenic mice.

Gene therapy of bone would benefit from the availability of vectors that provide stable, osteoblast-specific expression. This would allow bone-specific expression of Col1a1 cDNAs for treatment of osteogenesis imperfecta. In addition, such a vector would restrict expression of secreted therapeutic proteins to the bone-synthesizing regions of the bone marrow after ex vivo transduction of marrow stromal cells and reintroduction of the cells into patients. Retrovirus vectors stably integrate into target cell genomes; however, long-term regulated expression from internal cellular promoters has not been consistently achieved. In some cases this is due to a stem cell-specific mechanism for transcriptional repression of retroviruses. We evaluated the ability of self-inactivating ROSA-derived vectors containing a bone-directed 2.3-kb rat Col1a1 promoter to display osteoblast-specific expression. In vitro expression was examined in bone marrow stromal cell cultures induced to undergo osteoblastic differentiation. In vivo expression was evaluated in chimeric mice derived from transduced embryonic stem cells. The results indicate that self-inactivating retrovirus vectors containing the Col1a1 promoter are not permanently inactivated in embryonic stem cells and are specifically expressed in osteoblasts in vivo and in vitro. Thus these vectors should be useful for bone-directed gene therapy.

Animals↗

PubMed2001

Human bone marrow stromal cells are efficiently transduced by vesicular stomatitis virus-pseudotyped retrovectors without affecting subsequent osteoblastic differentiation.

This study tested the transduction efficiency of human bone marrow stromal cells (hBMSCs) with vesicular stomatitis virus (VSV)-pseudotyped retrovectors and their subsequent osteogenic differentiation in vitro. Two different retrovectors encoding beta-galactosidase (beta-gal) or enhanced green fluorescent protein (eGFP) as marker genes were examined for transduction of hBMSCs. hBMSCs were obtained from bone marrow filtrates of normal donors (aged 5-35 years), cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal calf serum and infected with retrovectors soon after the adherent cells started to form individual colonies. Transduced hBMSCs were observed to express eGFP protein 4-7 days after infection in primary cultures, and the majority of hBMSCs were eGFP-positive. hBMSCs were also stained for beta-gal in the secondary cultures and virtually all hBMSCs expressed beta-gal activity. Transduced hBMSCs were examined for their osteogenic potential. These cells were found to express markers of osteogenic differentiation, including alkaline phosphatase, type I collagen, bone sialoprotein, decorin, and osteocalcin, as strongly as uninfected control cells. Mineralization was also induced by dexamethasone in transduced cells as well as control cells. These results demonstrate that hBMSCs are highly susceptible to infection with VSV-pseudotyped retrovectors with the majority of cultured cells expressing the viral transgenes without antibiotic selection. Transduced cells retain their osteogenic potential in vitro. hBMSCs are a promising cellular vehicle for systemic human gene therapy and VSV-pseudotyped retrovectors should be effective for their in vitro transduction prior to cellular engraftment.

Adolescent↗

PubMed2001

Reduction of target gene expression by a modified U1 snRNA.

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

3T3 Cells↗

PubMed2001

Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice.

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Alkaline Phosphatase↗

PubMed2001

Col1a1-driven transgenic markers of osteoblast lineage progression.

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.

Animals↗

PubMed2001

Dlx5 induces expression of COL1A1 promoter contained in a retrovirus vector.

AIM: To determine whether retrovirally expressed Dlx5, a homeobox-containing transcription factor, can induce a 2.3 kb rat COL1A1 promoter-reporter construct, which is transduced into osteoblastic cells by the use of a retrovirus vector. METHODS: A self-inactivating retrovirus vector containing the rat COL1A1 driving green fluorescent protein (GFP) was transduced into chick calvarial periosteal cells. These cells were then infected with a replication-competent retroviral vector expressing Dlx5, or a control vector. The cells were cultured in the presence of ascorbic acid and beta-glycerol-phosphate, which promotes osteoblastic differentiation. Expression of the COL1A1 promoter was assessed by detecting GFP with fluorescence microscopy. RESULTS: GFP was detected only in cells infected with the Dlx5 expressing retrovirus. The GFP positive cells were observed in regions of the culture that had undergone osteoblastic differentiation, as detected by cell morphology and the presence of a mineralized matrix. CONCLUSION: The 2.3 kb rat COL1A1 promoter fragment contains elements responsive to Dlx5, and the self-inactivating retroviral vector allows these elements to be used appropriately.

Animals↗

PubMed2000

Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMP-mediated pathway in mouse calvariae.

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.

Animals↗

PubMed1999

Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation.

Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.

Animals↗

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

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.

Animals↗

PubMed1998

DNase I hypersensitive sites in the chromatin of the chicken Msx2 gene differ in anterior and posterior limb mesenchyme, calvarial osteoblasts and embryonic fibroblasts.

To identify candidate cis-acting regulatory regions involved in regulation of the Msx2 gene in anterior limb mesenchyme and calvarial osteoblasts, DNase I hypersensitive sites (DHSs) from -6.1 kb to +8.4 kb relative to the translation start site of the chicken Msx2 gene were identified in anterior and posterior limb mesenchyme, calvarial osteoblasts, and embryonic fibroblasts. A total of 12 DHSs were detected. Except for the DHS in the basal promoter region, none of the other DHSs were present in all four tissues, suggesting that the chromatin structure in the Msx2 gene locus is differently organized in these four cell types. One DHS was unique to Msx2 expressing cells and a second site to nonexpressing cells. Anterior and posterior limb mesenchyme had similar patterns of DHSs that were much more complex than observed in calvarial osteoblasts possibly reflecting differences in the complexity of Msx2 regulation in these two tissues.

Animals↗

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↗

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

A collagen enhancer-promoter construct in transgenic mice is markedly stimulated by ethanol administration.

Type I collagen synthesis and deposition is generally indicative of irreversible damage in alcohol-induced cirrhosis in humans. However, in rodents, ethanol alone does not readily cause hepatic fibrosis. To determine whether this is because of a lack of ethanol-responsive elements, an artificial enhancer construct controlling rat type I collagen gene transcription was prepared in transgenic mice. The gene construct, ColCAT3.6, was a chimeric sequence containing the marker chloramphenicol acetyltransferase (CAT) gene linked to 3.5 kb of the rat alpha 1(I) 5'-flanking DNA, and 115 base pairs (bp) of transcribed collagen gene. Groups of transgenic mice were given 4 g/kg ethanol orally, twice daily for 4 weeks. As a positive control for hepatic fibrosis, transgenic mice were given intraperitoneal injections of CCl4, twice weekly for 4 weeks. Livers were assayed for CAT activity. Endogenous mouse collagen alpha 1(I) messenger RNA (mRNA) and transgene CAT mRNA were measured by RNase protection assays. Collagen synthesis in livers from the transgenic mice treated with ethanol were increased over controls, but the levels were not significantly different. Endogenous collagen alpha 1(I) steady-state mRNA levels in ethanol-treated mice were not significantly different compared with saline-treated controls. However, the transgene mRNA levels in ethanol-treated animals increased approximately 21-fold compared with saline-treated controls, as measured by RNase protection assays. Furthermore, the transgene product as measured by CAT activity in ethanol-treated mice was significantly increased threefold over saline-treated controls. We conclude that the 5'-flanking region of the rat alpha 1(I) collagen gene does contain regulatory elements that are strongly responsive to ethanol administration.

Animals↗