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Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

Weston AD, Rosen V, Chandraratna RA, Underhill TM - J. Cell Biol. (2000)

Bottom Line: Underhill. 1997.These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation.Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biology, School of Dentistry, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

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The RARα-selective antagonist AGN 194301 inhibits stimulation of an RARE-reporter gene by all-trans RA in a dose-dependent manner. Low concentrations of AGN 194301 selectively inhibit RARα-mediated transactivation of the reporter gene in cultures cotransfected with an RARα expression construct. At higher concentrations of AGN 194301, the compound exhibits less selectivity, in that there is also decreased reporter gene activity in cultures cotransfected with RARβ or RARγ expression plasmids.
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Figure 4: The RARα-selective antagonist AGN 194301 inhibits stimulation of an RARE-reporter gene by all-trans RA in a dose-dependent manner. Low concentrations of AGN 194301 selectively inhibit RARα-mediated transactivation of the reporter gene in cultures cotransfected with an RARα expression construct. At higher concentrations of AGN 194301, the compound exhibits less selectivity, in that there is also decreased reporter gene activity in cultures cotransfected with RARβ or RARγ expression plasmids.

Mentions: We have demonstrated that continued RARα activity inhibits the transition of prechondrogenic cells to chondroblasts. Consequently, we would expect that the abrogation of RARα activity would stimulate chondroblast differentiation and/or cartilage formation. RAR activity as used herein refers to the level of RA-induced transcriptional activity of the RARs. To examine the possibility that inhibition of RARα activity stimulates cartilage formation, we treated wild-type cultures with the RARα-specific antagonist AGN 194301 (Teng et al. 1997). At the concentrations of AGN 194301 (1 μM) used in these experiments, RARα signaling was inhibited to ∼0.3% of 100 nM all trans-RA–treated controls, while RARβ and RARγ were inhibited to ∼18% and ∼26% of controls, respectively (Fig. 4). Addition of 1 μM AGN 194301 to wild-type cultures lead to a dramatic increase in nodule number as compared with untreated control cultures (Fig. 5g, Fig. j, and Fig. m). After 8 d in culture there were 60% more nodules in antagonist treated cultures than in untreated cultures (Fig. 5 m). Previous studies showed that treatment of micromass cultures with anti-sense oligonucleotides to the RARs increased cartilage nodule number (Motoyama and Eto 1994; Jiang et al. 1995), however, an RAR inhibitor to all three RARs had no effect on cartilage nodule formation (Kochhar et al. 1998). Most of the chondrogenic stimulatory properties of the antagonist appear to be mediated through inhibition of RARα, however, it cannot be entirely discounted that diminution of RARβ or RARγ signaling may have contributed to these results. Nonetheless, loss of RAR activity stimulates cartilage formation while continued RAR activity inhibits cartilage formation.


Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

Weston AD, Rosen V, Chandraratna RA, Underhill TM - J. Cell Biol. (2000)

The RARα-selective antagonist AGN 194301 inhibits stimulation of an RARE-reporter gene by all-trans RA in a dose-dependent manner. Low concentrations of AGN 194301 selectively inhibit RARα-mediated transactivation of the reporter gene in cultures cotransfected with an RARα expression construct. At higher concentrations of AGN 194301, the compound exhibits less selectivity, in that there is also decreased reporter gene activity in cultures cotransfected with RARβ or RARγ expression plasmids.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169377&req=5

Figure 4: The RARα-selective antagonist AGN 194301 inhibits stimulation of an RARE-reporter gene by all-trans RA in a dose-dependent manner. Low concentrations of AGN 194301 selectively inhibit RARα-mediated transactivation of the reporter gene in cultures cotransfected with an RARα expression construct. At higher concentrations of AGN 194301, the compound exhibits less selectivity, in that there is also decreased reporter gene activity in cultures cotransfected with RARβ or RARγ expression plasmids.
Mentions: We have demonstrated that continued RARα activity inhibits the transition of prechondrogenic cells to chondroblasts. Consequently, we would expect that the abrogation of RARα activity would stimulate chondroblast differentiation and/or cartilage formation. RAR activity as used herein refers to the level of RA-induced transcriptional activity of the RARs. To examine the possibility that inhibition of RARα activity stimulates cartilage formation, we treated wild-type cultures with the RARα-specific antagonist AGN 194301 (Teng et al. 1997). At the concentrations of AGN 194301 (1 μM) used in these experiments, RARα signaling was inhibited to ∼0.3% of 100 nM all trans-RA–treated controls, while RARβ and RARγ were inhibited to ∼18% and ∼26% of controls, respectively (Fig. 4). Addition of 1 μM AGN 194301 to wild-type cultures lead to a dramatic increase in nodule number as compared with untreated control cultures (Fig. 5g, Fig. j, and Fig. m). After 8 d in culture there were 60% more nodules in antagonist treated cultures than in untreated cultures (Fig. 5 m). Previous studies showed that treatment of micromass cultures with anti-sense oligonucleotides to the RARs increased cartilage nodule number (Motoyama and Eto 1994; Jiang et al. 1995), however, an RAR inhibitor to all three RARs had no effect on cartilage nodule formation (Kochhar et al. 1998). Most of the chondrogenic stimulatory properties of the antagonist appear to be mediated through inhibition of RARα, however, it cannot be entirely discounted that diminution of RARβ or RARγ signaling may have contributed to these results. Nonetheless, loss of RAR activity stimulates cartilage formation while continued RAR activity inhibits cartilage formation.

Bottom Line: Underhill. 1997.These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation.Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biology, School of Dentistry, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

Show MeSH
Related in: MedlinePlus