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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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Collagenase-3 expression in metatarsal bone rudiments from embryos with targeted deletion of the Cbfa1 and MT1-MMP genes. Cbfa1- (a–d) and MT1-MMP– (e–h) metatarsal rudiments were untreated (a, b, e, and f) or treated with RA (c, d, g, and h) and stained with Alcian blue and nuclear fast red (a, c, e, and g) or hybridized with a labeled antisense riboprobe for collagenase-3 (b, d, f, and h). Untreated Cbfa1−/− rudiments showed signs of cellular disorganization, especially in the hypertrophic zone where a clear Alcian blue staining was observed (a). Treatment of Cbfa1−/− metatarsi with RA resulted in a marked inhibition of the chondrocytic hypertrophy (c). In situ hybridization studies failed to find positive signal for collagenase-3 in either untreated (b) or RA-treated (d) rudiments. Untreated MT1-MMP−/− rudiments showed a disorganized hypertrophic zone with an intense Alcian blue staining (e). Collagenase-3 expression was low but could be observed in cells located mainly in the perichondrium (f). Treatment of MT1-MMP−/− metatarsi with RA induced some cytological changes (g) and resulted in an increased collagenase-3 expression (h) at the hypertrophic chondrocytes. Bars, 200 μm.
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fig4: Collagenase-3 expression in metatarsal bone rudiments from embryos with targeted deletion of the Cbfa1 and MT1-MMP genes. Cbfa1- (a–d) and MT1-MMP– (e–h) metatarsal rudiments were untreated (a, b, e, and f) or treated with RA (c, d, g, and h) and stained with Alcian blue and nuclear fast red (a, c, e, and g) or hybridized with a labeled antisense riboprobe for collagenase-3 (b, d, f, and h). Untreated Cbfa1−/− rudiments showed signs of cellular disorganization, especially in the hypertrophic zone where a clear Alcian blue staining was observed (a). Treatment of Cbfa1−/− metatarsi with RA resulted in a marked inhibition of the chondrocytic hypertrophy (c). In situ hybridization studies failed to find positive signal for collagenase-3 in either untreated (b) or RA-treated (d) rudiments. Untreated MT1-MMP−/− rudiments showed a disorganized hypertrophic zone with an intense Alcian blue staining (e). Collagenase-3 expression was low but could be observed in cells located mainly in the perichondrium (f). Treatment of MT1-MMP−/− metatarsi with RA induced some cytological changes (g) and resulted in an increased collagenase-3 expression (h) at the hypertrophic chondrocytes. Bars, 200 μm.

Mentions: To further support the possibility that the effect of RA on collagenase-3 expression was mediated by Cbfa1, we performed metatarsal organ cultures from embryos with targeted deletion of the Cbfa1 gene. A comparative morphological analysis with control littermate metatarsal rudiments revealed that Cbfa1- rudiments were shorter in both total length (3.0 ± 0.11 mm, n = 4 versus 3.7 ± 0.14 mm, n = 5, p < 0.01) and fraction of hypertrophic cartilage. RA treatment of rudiments from Cbfa1−/− mice resulted in a slight decrease of longitudinal growth (2.7 ± 0.12 mm, n = 4 versus 3.0 ± 0.11 mm, n = 4, p < 0.01) that was proportionally lower than that observed in RA-treated Cbfa1+/+ rudiments (2.8 ± 0.14 mm, n = 5 versus 3.7 ± 0.14 mm, n = 5, p < 0.01). RA treatment of Cbfa1−/− rudiments also resulted in a marked inhibition of chondrocytic hypertrophy (Fig. 4, a and c), a result similar to that observed in wild-type samples (Fig. 1). However, RA treatment did not induce any increase of perichondrial thickness in Cbfa1- metatarsal rudiments, a result different from that observed in the wild-type samples. In situ hybridization studies showed that collagenase-3 expression was absent in both untreated and RA-treated Cbfa1−/− metatarsal bone rudiments (Fig. 4, b and d), which agrees with the proposal that Cbfa1 is required for the RA-induced increase of collagenase-3 expression. We next examined the possibility that the effect of RA on collagenase-3 expression could be influenced by the absence of MT1-MMP, which as mentioned above is involved in both bone formation and collagenase-3 activation. To this end, we used metatarsal cultures from MT1-MMP– embryos and analyzed the effects of RA on these explants. Similar to the case of Cbfa1- rudiments, MT1-MMP−/− rudiments were shorter than wild-type littermate samples (2.8 ± 0.13 mm, n = 6, versus 3.5 ± 0.16 mm, n = 4, p < 0.01) and showed a clear histological alteration at the hypertrophic zone where chondrocytes were smaller in size and appeared highly disorganized (Fig. 4 e). RA treatment of MT1-MMP−/− rudiments induced a significant decrease in length (2.1 ± 0.18 mm, n = 6 versus 2.8 ± 0.13 mm, n = 6, p < 0.01), which was comparable to that observed in wild-type rudiments (2.9 ± 0.13 mm, n = 4 versus 3.5 ± 0.16 mm, n = 4, p < 0.01). The observed cytological alterations in untreated MT1-MMP−/− samples were also enhanced in RA-treated rudiments (Fig. 4 g). In situ hybridization studies showed positive collagenase-3 expression in both untreated and RA-treated MT1-MMP−/− metatarsi (Figs. 4, f and h). In untreated rudiments, labeling was restricted to some hypertrophic chondrocytes and cells of the perichondrium, a pattern similar to that observed in samples from control mice (Fig. 4 f). Likewise, RA-treatment of MT1-MMP−/− rudiments resulted in an increased expression of collagenase-3. However, in these mutant bone rudiments collagenase-3 expression was found mainly at the disorganized hypertrophic chondrocytes located at the middle of the bone (Fig. 4 h). Like in Cbfa1−/− metatarsi, RA treatment did not induce any perichondrial expansion in MT1-MMP−/− rudiments. According to these results, we conclude that the RA-induced increase of collagenase-3 expression was not dependent of MT1-MMP.


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)

Collagenase-3 expression in metatarsal bone rudiments from embryos with targeted deletion of the Cbfa1 and MT1-MMP genes. Cbfa1- (a–d) and MT1-MMP– (e–h) metatarsal rudiments were untreated (a, b, e, and f) or treated with RA (c, d, g, and h) and stained with Alcian blue and nuclear fast red (a, c, e, and g) or hybridized with a labeled antisense riboprobe for collagenase-3 (b, d, f, and h). Untreated Cbfa1−/− rudiments showed signs of cellular disorganization, especially in the hypertrophic zone where a clear Alcian blue staining was observed (a). Treatment of Cbfa1−/− metatarsi with RA resulted in a marked inhibition of the chondrocytic hypertrophy (c). In situ hybridization studies failed to find positive signal for collagenase-3 in either untreated (b) or RA-treated (d) rudiments. Untreated MT1-MMP−/− rudiments showed a disorganized hypertrophic zone with an intense Alcian blue staining (e). Collagenase-3 expression was low but could be observed in cells located mainly in the perichondrium (f). Treatment of MT1-MMP−/− metatarsi with RA induced some cytological changes (g) and resulted in an increased collagenase-3 expression (h) at the hypertrophic chondrocytes. Bars, 200 μm.
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Related In: Results  -  Collection

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fig4: Collagenase-3 expression in metatarsal bone rudiments from embryos with targeted deletion of the Cbfa1 and MT1-MMP genes. Cbfa1- (a–d) and MT1-MMP– (e–h) metatarsal rudiments were untreated (a, b, e, and f) or treated with RA (c, d, g, and h) and stained with Alcian blue and nuclear fast red (a, c, e, and g) or hybridized with a labeled antisense riboprobe for collagenase-3 (b, d, f, and h). Untreated Cbfa1−/− rudiments showed signs of cellular disorganization, especially in the hypertrophic zone where a clear Alcian blue staining was observed (a). Treatment of Cbfa1−/− metatarsi with RA resulted in a marked inhibition of the chondrocytic hypertrophy (c). In situ hybridization studies failed to find positive signal for collagenase-3 in either untreated (b) or RA-treated (d) rudiments. Untreated MT1-MMP−/− rudiments showed a disorganized hypertrophic zone with an intense Alcian blue staining (e). Collagenase-3 expression was low but could be observed in cells located mainly in the perichondrium (f). Treatment of MT1-MMP−/− metatarsi with RA induced some cytological changes (g) and resulted in an increased collagenase-3 expression (h) at the hypertrophic chondrocytes. Bars, 200 μm.
Mentions: To further support the possibility that the effect of RA on collagenase-3 expression was mediated by Cbfa1, we performed metatarsal organ cultures from embryos with targeted deletion of the Cbfa1 gene. A comparative morphological analysis with control littermate metatarsal rudiments revealed that Cbfa1- rudiments were shorter in both total length (3.0 ± 0.11 mm, n = 4 versus 3.7 ± 0.14 mm, n = 5, p < 0.01) and fraction of hypertrophic cartilage. RA treatment of rudiments from Cbfa1−/− mice resulted in a slight decrease of longitudinal growth (2.7 ± 0.12 mm, n = 4 versus 3.0 ± 0.11 mm, n = 4, p < 0.01) that was proportionally lower than that observed in RA-treated Cbfa1+/+ rudiments (2.8 ± 0.14 mm, n = 5 versus 3.7 ± 0.14 mm, n = 5, p < 0.01). RA treatment of Cbfa1−/− rudiments also resulted in a marked inhibition of chondrocytic hypertrophy (Fig. 4, a and c), a result similar to that observed in wild-type samples (Fig. 1). However, RA treatment did not induce any increase of perichondrial thickness in Cbfa1- metatarsal rudiments, a result different from that observed in the wild-type samples. In situ hybridization studies showed that collagenase-3 expression was absent in both untreated and RA-treated Cbfa1−/− metatarsal bone rudiments (Fig. 4, b and d), which agrees with the proposal that Cbfa1 is required for the RA-induced increase of collagenase-3 expression. We next examined the possibility that the effect of RA on collagenase-3 expression could be influenced by the absence of MT1-MMP, which as mentioned above is involved in both bone formation and collagenase-3 activation. To this end, we used metatarsal cultures from MT1-MMP– embryos and analyzed the effects of RA on these explants. Similar to the case of Cbfa1- rudiments, MT1-MMP−/− rudiments were shorter than wild-type littermate samples (2.8 ± 0.13 mm, n = 6, versus 3.5 ± 0.16 mm, n = 4, p < 0.01) and showed a clear histological alteration at the hypertrophic zone where chondrocytes were smaller in size and appeared highly disorganized (Fig. 4 e). RA treatment of MT1-MMP−/− rudiments induced a significant decrease in length (2.1 ± 0.18 mm, n = 6 versus 2.8 ± 0.13 mm, n = 6, p < 0.01), which was comparable to that observed in wild-type rudiments (2.9 ± 0.13 mm, n = 4 versus 3.5 ± 0.16 mm, n = 4, p < 0.01). The observed cytological alterations in untreated MT1-MMP−/− samples were also enhanced in RA-treated rudiments (Fig. 4 g). In situ hybridization studies showed positive collagenase-3 expression in both untreated and RA-treated MT1-MMP−/− metatarsi (Figs. 4, f and h). In untreated rudiments, labeling was restricted to some hypertrophic chondrocytes and cells of the perichondrium, a pattern similar to that observed in samples from control mice (Fig. 4 f). Likewise, RA-treatment of MT1-MMP−/− rudiments resulted in an increased expression of collagenase-3. However, in these mutant bone rudiments collagenase-3 expression was found mainly at the disorganized hypertrophic chondrocytes located at the middle of the bone (Fig. 4 h). Like in Cbfa1−/− metatarsi, RA treatment did not induce any perichondrial expansion in MT1-MMP−/− rudiments. According to these results, we conclude that the RA-induced increase of collagenase-3 expression was not dependent of MT1-MMP.

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