Limits...
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.

Show MeSH

Related in: MedlinePlus

Map of the MT-DNIIR expression plasmid. The  EcoRI/XbaI fragment of the human TGF-β type II receptor from  the plasmid p102 containing a FLAG epitope tag and the signal  sequence (SP), ligand binding, transmembrane (TM), and juxtamembrane (JM) domains of the receptor was inserted into the  BamHI site of the MT-β metallothionein expression vector by  blunt end ligation. The MT-β vector contains four metal responsive elements and the β globin TATA element, splice sites, and  polyadenylation signal (69). The HindIII/BglI fragment was injected into single cell embryos. Arrows mark the location of  primer sequences used for PCR and RT-PCR analysis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139797&req=5

Figure 1: Map of the MT-DNIIR expression plasmid. The EcoRI/XbaI fragment of the human TGF-β type II receptor from the plasmid p102 containing a FLAG epitope tag and the signal sequence (SP), ligand binding, transmembrane (TM), and juxtamembrane (JM) domains of the receptor was inserted into the BamHI site of the MT-β metallothionein expression vector by blunt end ligation. The MT-β vector contains four metal responsive elements and the β globin TATA element, splice sites, and polyadenylation signal (69). The HindIII/BglI fragment was injected into single cell embryos. Arrows mark the location of primer sequences used for PCR and RT-PCR analysis.

Mentions: The MT-DNIIR expression plasmid was used to generate transgenic mice (see Fig. 1). The EcoRI/XbaI fragment of p102 containing the truncated human TGF-β type II receptor (11) was inserted into the BamHI site of MT-β (78) by blunt end ligation. The HindIII/BglI fragment containing the transgene under the metallothionein promoter was microinjected into the pronuclei of single cell embyos from crosses of C57BL/6 and DBA mice (26). Mice were maintained on Purina mouse chow and tap water. Transgenic mice were identified by Southern blot (59) and PCR analyses of genomic DNA isolated from mouse tails by proteinase K digestion and phenol/chloroform extraction. For Southern blots, genomic DNA was digested with PstI/EcoRI and the BamHI/EcoRI rabbit β-globin fragment from MT-β was used as the probe. PCR was performed using primers to the FLAG epitope sequence: ATC GTC ATC GTC TTT GTA GTC and human TGF-β type II receptor: TCC CAC CGC ACG TTC AGA AG. Genomic DNA was amplified for 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s, and elongation for 2 min at 72°C in reaction buffer containing 2 mM MgCl, 1× PCR buffer (Perkin Elmer, Blanchburg, NJ), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), and 0.2 μM of each primer.


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)

Map of the MT-DNIIR expression plasmid. The  EcoRI/XbaI fragment of the human TGF-β type II receptor from  the plasmid p102 containing a FLAG epitope tag and the signal  sequence (SP), ligand binding, transmembrane (TM), and juxtamembrane (JM) domains of the receptor was inserted into the  BamHI site of the MT-β metallothionein expression vector by  blunt end ligation. The MT-β vector contains four metal responsive elements and the β globin TATA element, splice sites, and  polyadenylation signal (69). The HindIII/BglI fragment was injected into single cell embryos. Arrows mark the location of  primer sequences used for PCR and RT-PCR analysis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Map of the MT-DNIIR expression plasmid. The EcoRI/XbaI fragment of the human TGF-β type II receptor from the plasmid p102 containing a FLAG epitope tag and the signal sequence (SP), ligand binding, transmembrane (TM), and juxtamembrane (JM) domains of the receptor was inserted into the BamHI site of the MT-β metallothionein expression vector by blunt end ligation. The MT-β vector contains four metal responsive elements and the β globin TATA element, splice sites, and polyadenylation signal (69). The HindIII/BglI fragment was injected into single cell embryos. Arrows mark the location of primer sequences used for PCR and RT-PCR analysis.
Mentions: The MT-DNIIR expression plasmid was used to generate transgenic mice (see Fig. 1). The EcoRI/XbaI fragment of p102 containing the truncated human TGF-β type II receptor (11) was inserted into the BamHI site of MT-β (78) by blunt end ligation. The HindIII/BglI fragment containing the transgene under the metallothionein promoter was microinjected into the pronuclei of single cell embyos from crosses of C57BL/6 and DBA mice (26). Mice were maintained on Purina mouse chow and tap water. Transgenic mice were identified by Southern blot (59) and PCR analyses of genomic DNA isolated from mouse tails by proteinase K digestion and phenol/chloroform extraction. For Southern blots, genomic DNA was digested with PstI/EcoRI and the BamHI/EcoRI rabbit β-globin fragment from MT-β was used as the probe. PCR was performed using primers to the FLAG epitope sequence: ATC GTC ATC GTC TTT GTA GTC and human TGF-β type II receptor: TCC CAC CGC ACG TTC AGA AG. Genomic DNA was amplified for 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s, and elongation for 2 min at 72°C in reaction buffer containing 2 mM MgCl, 1× PCR buffer (Perkin Elmer, Blanchburg, NJ), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), and 0.2 μM of each primer.

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