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A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation.

Jiménez MJ, Balbín M, Alvarez J, Komori T, Bianco P, Holmbeck K, Birkedal-Hansen H, López JM, López-Otín C - J. Cell Biol. (2001)

Bottom Line: We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells.These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment.RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain.

ABSTRACT
Tissue-remodeling processes are largely mediated by members of the matrix metalloproteinase (MMP) family of endopeptidases whose expression is strictly controlled both spatially and temporally. In this article, we have examined the molecular mechanisms that could contribute to modulate the expression of MMPs like collagenase-3 and MT1-MMP during bone formation. We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells. This effect is dose and time dependent, requires the de novo synthesis of proteins, and is mediated by RAR-RXR heterodimers. Analysis of the signal transduction mechanisms underlying the upregulating effect of RA on collagenase-3 expression demonstrated that this factor acts through a signaling pathway involving p38 mitogen-activated protein kinase. RA treatment of chondrocytic cells also induces the production of MT1-MMP, a membrane-bound metalloproteinase essential for skeletal formation, which participates in a proteolytic cascade with collagenase-3. The production of these MMPs is concomitant with the development of an RA-induced differentiation program characterized by formation of a mineralized bone matrix, downregulation of chondrocyte markers like type II collagen, and upregulation of osteoblastic markers such as osteocalcin. These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment. RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells. The dynamics of Cbfa1, MMPs, and osteocalcin expression is consistent with the fact that these genes could be part of a regulatory cascade initiated by RA and leading to the induction of Cbfa1, which in turn would upregulate the expression of some of their target genes like collagenase-3 and osteocalcin.

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Effect of different RA receptors agonists and antagonists on collagenase-3 expression. RCS cells were cultured for 48 h in the presence of 10−6 M of different agonists (Ro40-6055, αRAR agonist; Ro48-2249, βRAR agonist; Ro44-4753, γRAR agonist; RXR agonist, LG100064) (a) or with 10−6 M RA plus different antagonists (Ro41-5253, αRAR antagonist; LE135, βRAR antagonist) (b). Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot.
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fig6: Effect of different RA receptors agonists and antagonists on collagenase-3 expression. RCS cells were cultured for 48 h in the presence of 10−6 M of different agonists (Ro40-6055, αRAR agonist; Ro48-2249, βRAR agonist; Ro44-4753, γRAR agonist; RXR agonist, LG100064) (a) or with 10−6 M RA plus different antagonists (Ro41-5253, αRAR antagonist; LE135, βRAR antagonist) (b). Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot.

Mentions: The above observation that all-trans RA upregulated collagenase-3 expression in chondrocytic cells prompted us to examine the nature of the nuclear retinoic acid receptors presumably involved in this process. To this purpose, we first analyzed by Northern blot the expression levels of the different retinoid receptors in RCS cells (Fig. 5). This analysis revealed that RARα and RARγ receptors are constitutively expressed but show some variations after RA treatment, indicating that both of them could be involved in the process. We next evaluated the effect of different agonistic and antagonistic retinoids on collagenase-3 expression in these cells. As shown in Fig. 6 a, the RARα-selective agonist Ro40-6055 induced a collagenase-3 expression similar to that observed with all-trans RA. In addition, when cells were incubated with the RARβ-selective (Ro48-2249) or the RARγ-selective (R044-4753) retinoids, collagenase-3 expression was also induced, although the upregulatory effect was much lower than that observed after treatment with RARα agonists. Furthermore, the RARα-selective antagonist Ro41-5253 extensively blocked the RA-induced accumulation of collagenase-3, whereas the RARβ-selective antagonist LE135 only showed a slight inhibitory effect (Fig. 6 b). Finally, we examined the possibility that RXR-mediated signaling pathways could also contribute to the observed upregulation of collagenase-3 expression by retinoids. Thus, RCS cells were incubated with different RAR agonists in the presence or absence of the RXR-selective retinoid LG100064, and collagenase-3 levels were analyzed by Northern blot. As shown in Fig. 6 a, the presence of this RXR-selective retinoid produced a significant increase in the collagenase-3 mRNA levels when compared with values obtained after incubation with RAR agonists alone. Taken together, these results indicate that RAR-RXR heterodimers, likely containing RARα isoforms, are involved in the transduction of the retinoid signal that leads to the induction of collagenase-3 in chondrocytic cells.


A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation.

Jiménez MJ, Balbín M, Alvarez J, Komori T, Bianco P, Holmbeck K, Birkedal-Hansen H, López JM, López-Otín C - J. Cell Biol. (2001)

Effect of different RA receptors agonists and antagonists on collagenase-3 expression. RCS cells were cultured for 48 h in the presence of 10−6 M of different agonists (Ro40-6055, αRAR agonist; Ro48-2249, βRAR agonist; Ro44-4753, γRAR agonist; RXR agonist, LG100064) (a) or with 10−6 M RA plus different antagonists (Ro41-5253, αRAR antagonist; LE135, βRAR antagonist) (b). Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199331&req=5

fig6: Effect of different RA receptors agonists and antagonists on collagenase-3 expression. RCS cells were cultured for 48 h in the presence of 10−6 M of different agonists (Ro40-6055, αRAR agonist; Ro48-2249, βRAR agonist; Ro44-4753, γRAR agonist; RXR agonist, LG100064) (a) or with 10−6 M RA plus different antagonists (Ro41-5253, αRAR antagonist; LE135, βRAR antagonist) (b). Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot.
Mentions: The above observation that all-trans RA upregulated collagenase-3 expression in chondrocytic cells prompted us to examine the nature of the nuclear retinoic acid receptors presumably involved in this process. To this purpose, we first analyzed by Northern blot the expression levels of the different retinoid receptors in RCS cells (Fig. 5). This analysis revealed that RARα and RARγ receptors are constitutively expressed but show some variations after RA treatment, indicating that both of them could be involved in the process. We next evaluated the effect of different agonistic and antagonistic retinoids on collagenase-3 expression in these cells. As shown in Fig. 6 a, the RARα-selective agonist Ro40-6055 induced a collagenase-3 expression similar to that observed with all-trans RA. In addition, when cells were incubated with the RARβ-selective (Ro48-2249) or the RARγ-selective (R044-4753) retinoids, collagenase-3 expression was also induced, although the upregulatory effect was much lower than that observed after treatment with RARα agonists. Furthermore, the RARα-selective antagonist Ro41-5253 extensively blocked the RA-induced accumulation of collagenase-3, whereas the RARβ-selective antagonist LE135 only showed a slight inhibitory effect (Fig. 6 b). Finally, we examined the possibility that RXR-mediated signaling pathways could also contribute to the observed upregulation of collagenase-3 expression by retinoids. Thus, RCS cells were incubated with different RAR agonists in the presence or absence of the RXR-selective retinoid LG100064, and collagenase-3 levels were analyzed by Northern blot. As shown in Fig. 6 a, the presence of this RXR-selective retinoid produced a significant increase in the collagenase-3 mRNA levels when compared with values obtained after incubation with RAR agonists alone. Taken together, these results indicate that RAR-RXR heterodimers, likely containing RARα isoforms, are involved in the transduction of the retinoid signal that leads to the induction of collagenase-3 in chondrocytic cells.

Bottom Line: We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells.These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment.RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain.

ABSTRACT
Tissue-remodeling processes are largely mediated by members of the matrix metalloproteinase (MMP) family of endopeptidases whose expression is strictly controlled both spatially and temporally. In this article, we have examined the molecular mechanisms that could contribute to modulate the expression of MMPs like collagenase-3 and MT1-MMP during bone formation. We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells. This effect is dose and time dependent, requires the de novo synthesis of proteins, and is mediated by RAR-RXR heterodimers. Analysis of the signal transduction mechanisms underlying the upregulating effect of RA on collagenase-3 expression demonstrated that this factor acts through a signaling pathway involving p38 mitogen-activated protein kinase. RA treatment of chondrocytic cells also induces the production of MT1-MMP, a membrane-bound metalloproteinase essential for skeletal formation, which participates in a proteolytic cascade with collagenase-3. The production of these MMPs is concomitant with the development of an RA-induced differentiation program characterized by formation of a mineralized bone matrix, downregulation of chondrocyte markers like type II collagen, and upregulation of osteoblastic markers such as osteocalcin. These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment. RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells. The dynamics of Cbfa1, MMPs, and osteocalcin expression is consistent with the fact that these genes could be part of a regulatory cascade initiated by RA and leading to the induction of Cbfa1, which in turn would upregulate the expression of some of their target genes like collagenase-3 and osteocalcin.

Show MeSH
Related in: MedlinePlus