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Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis.

Serra R, Johnson M, Filvaroff EH, LaBorde J, Sheehan DM, Derynck R, Moses HL - J. Cell Biol. (1997)

Bottom Line: Lower levels of DNIIR mRNA were detected in growth plate cartilage.It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation.The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and the Vanderbilt Cancer Center, Vanderbilt University, Nashville, Tennessee 37232, USA.

ABSTRACT
Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

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RT-PCR analysis of DNIIR mRNA expression in MT-DNIIR transgenic mouse lines. RNA isolated and pooled from  the hind limbs of two to four wild-type (WT) or transgenic (MT-DNIIR-4, -15, -27, -28, -30) mice maintained on normal food and  tap water was analyzed by RT-PCR. To specifically amplify the  truncated DNIIR cDNA, primers targeted to FLAG epitope sequences were used (Fig. 1). Amplifications of GAPDH was used  as an internal control. Two separate assays are shown separated  by the black bar.
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Figure 3: RT-PCR analysis of DNIIR mRNA expression in MT-DNIIR transgenic mouse lines. RNA isolated and pooled from the hind limbs of two to four wild-type (WT) or transgenic (MT-DNIIR-4, -15, -27, -28, -30) mice maintained on normal food and tap water was analyzed by RT-PCR. To specifically amplify the truncated DNIIR cDNA, primers targeted to FLAG epitope sequences were used (Fig. 1). Amplifications of GAPDH was used as an internal control. Two separate assays are shown separated by the black bar.

Mentions: Since transgenic mice developed skeletal defects in the absence of exogenously added zinc, we sought to determine if expression of the transgene in the MT-DNIIR-4 and -27 lines correlated with the skeletal phenotype and if the hybrid promoter allowed leaky expression in the absence of zinc. We thus prepared adult skeletal mRNA from hind limbs of mice maintained on normal food and tap water and examined using RT-PCR analysis whether DNIIR mRNA was expressed in transgenic (MTR4, -15, -27, -28, -30) and wild-type mice (Fig. 3). Transgene-specific (DNIIR) primers were used to amplify mutant but not endogenous type II receptor cDNA sequences. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize for the amount of cDNA used in each reaction. As shown in Fig. 3, DNIIR mRNA was expressed in skeletal tissue from MT-DNIIR-4 and -27 mice in the absence of exogenously added zinc. DNIIR mRNA was not detected in MT-DNIIR-27 mice that did not exhibit skeletal defects (data not shown). There was very little basal expression of the DNIIR transgene in skeletal tissue from MT-DNIIR-15, -28, and -30 mice. We have also characterized DNIIR mRNA expression in other tissues in each of the MT-DNIIR mouse lines (data not shown). Our analyses showed that DNIIR mRNA was expressed in different sets of tissues depending on the individual MT-DNIIR mouse line. This heterogeneity in expression pattern is most likely due to the presence of only minimal gene regulatory elements in this version of the metallothionin promoter, which may make its transcriptional activity sensitive to DNA surrounding the transgene integration site. However, the skeletal phenotype is most likely not due to disruption of an unknown gene for several reasons. First, the skeletal phenotype is observed in heterozygous mice. Second, two separate lines of mice demonstrated a similar skeletal defect and, third, the sites of constituative expression of the DNIIR mRNA correlated with skeletal abnormalities. All of our experiments outlined below were performed with heterozygous MT-DNIIR-4 mice maintained on normal food and tap water.


Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis.

Serra R, Johnson M, Filvaroff EH, LaBorde J, Sheehan DM, Derynck R, Moses HL - J. Cell Biol. (1997)

RT-PCR analysis of DNIIR mRNA expression in MT-DNIIR transgenic mouse lines. RNA isolated and pooled from  the hind limbs of two to four wild-type (WT) or transgenic (MT-DNIIR-4, -15, -27, -28, -30) mice maintained on normal food and  tap water was analyzed by RT-PCR. To specifically amplify the  truncated DNIIR cDNA, primers targeted to FLAG epitope sequences were used (Fig. 1). Amplifications of GAPDH was used  as an internal control. Two separate assays are shown separated  by the black bar.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139797&req=5

Figure 3: RT-PCR analysis of DNIIR mRNA expression in MT-DNIIR transgenic mouse lines. RNA isolated and pooled from the hind limbs of two to four wild-type (WT) or transgenic (MT-DNIIR-4, -15, -27, -28, -30) mice maintained on normal food and tap water was analyzed by RT-PCR. To specifically amplify the truncated DNIIR cDNA, primers targeted to FLAG epitope sequences were used (Fig. 1). Amplifications of GAPDH was used as an internal control. Two separate assays are shown separated by the black bar.
Mentions: Since transgenic mice developed skeletal defects in the absence of exogenously added zinc, we sought to determine if expression of the transgene in the MT-DNIIR-4 and -27 lines correlated with the skeletal phenotype and if the hybrid promoter allowed leaky expression in the absence of zinc. We thus prepared adult skeletal mRNA from hind limbs of mice maintained on normal food and tap water and examined using RT-PCR analysis whether DNIIR mRNA was expressed in transgenic (MTR4, -15, -27, -28, -30) and wild-type mice (Fig. 3). Transgene-specific (DNIIR) primers were used to amplify mutant but not endogenous type II receptor cDNA sequences. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize for the amount of cDNA used in each reaction. As shown in Fig. 3, DNIIR mRNA was expressed in skeletal tissue from MT-DNIIR-4 and -27 mice in the absence of exogenously added zinc. DNIIR mRNA was not detected in MT-DNIIR-27 mice that did not exhibit skeletal defects (data not shown). There was very little basal expression of the DNIIR transgene in skeletal tissue from MT-DNIIR-15, -28, and -30 mice. We have also characterized DNIIR mRNA expression in other tissues in each of the MT-DNIIR mouse lines (data not shown). Our analyses showed that DNIIR mRNA was expressed in different sets of tissues depending on the individual MT-DNIIR mouse line. This heterogeneity in expression pattern is most likely due to the presence of only minimal gene regulatory elements in this version of the metallothionin promoter, which may make its transcriptional activity sensitive to DNA surrounding the transgene integration site. However, the skeletal phenotype is most likely not due to disruption of an unknown gene for several reasons. First, the skeletal phenotype is observed in heterozygous mice. Second, two separate lines of mice demonstrated a similar skeletal defect and, third, the sites of constituative expression of the DNIIR mRNA correlated with skeletal abnormalities. All of our experiments outlined below were performed with heterozygous MT-DNIIR-4 mice maintained on normal food and tap water.

Bottom Line: Lower levels of DNIIR mRNA were detected in growth plate cartilage.It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation.The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and the Vanderbilt Cancer Center, Vanderbilt University, Nashville, Tennessee 37232, USA.

ABSTRACT
Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

Show MeSH
Related in: MedlinePlus