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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. Paraffin sections from embryonic metatarsal rudiments untreated (a and c) or treated with RA (b and d) were hybridized with a labeled antisense riboprobe for collagenase-3. In untreated cultures, collagenase-3 expression was relatively low, being restricted to a low number of cells located mainly in the perichondrium. (c) Higher magnification of a peripheric area showing that most collagenase-3–positive cells were small sized and flat shaped. Metatarsals treated with RA (b) showed higher levels of collagenase-3 expression. Positive signal was increased in zones in which the perichondrium invaded into the underlying cartilage (d). (e–h) Metatarsal sections untreated (e) and treated with RA (f–h) and stained with toluidine blue (e, f, and h) and Alcian blue (g). A clear boundary between proliferating and hypertrophic zones was observed in untreated metatarsals (e). RA-treated cultures showed a partial inhibition of cellular enlargement during chondrocytic hypertrophy resulting in ill-defined boundaries between proliferating and hypertrophic zones (f). Invasion of cartilage by cells from the perichondrium also increased in RA-treated cultures (f, arrowhead; g and h). Weakly Alcian blue–stained intrachondral cells invading from the perichondrium are observed in g. (h) Higher magnification of intrachondral cells showed in g and stained with toluidine blue. These cells are elongated and have basophilic cytoplasm. Bars: (a, e, and f) 100 μm; (b) 160 μm; (c, d, and g) 40 μm; (h) 25 μm.
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fig1: Collagenase-3 expression in metatarsal bone rudiments. Paraffin sections from embryonic metatarsal rudiments untreated (a and c) or treated with RA (b and d) were hybridized with a labeled antisense riboprobe for collagenase-3. In untreated cultures, collagenase-3 expression was relatively low, being restricted to a low number of cells located mainly in the perichondrium. (c) Higher magnification of a peripheric area showing that most collagenase-3–positive cells were small sized and flat shaped. Metatarsals treated with RA (b) showed higher levels of collagenase-3 expression. Positive signal was increased in zones in which the perichondrium invaded into the underlying cartilage (d). (e–h) Metatarsal sections untreated (e) and treated with RA (f–h) and stained with toluidine blue (e, f, and h) and Alcian blue (g). A clear boundary between proliferating and hypertrophic zones was observed in untreated metatarsals (e). RA-treated cultures showed a partial inhibition of cellular enlargement during chondrocytic hypertrophy resulting in ill-defined boundaries between proliferating and hypertrophic zones (f). Invasion of cartilage by cells from the perichondrium also increased in RA-treated cultures (f, arrowhead; g and h). Weakly Alcian blue–stained intrachondral cells invading from the perichondrium are observed in g. (h) Higher magnification of intrachondral cells showed in g and stained with toluidine blue. These cells are elongated and have basophilic cytoplasm. Bars: (a, e, and f) 100 μm; (b) 160 μm; (c, d, and g) 40 μm; (h) 25 μm.

Mentions: To study the putative effect of RA on the expression of collagenase-3 during bone formation, we first performed in situ hybridization experiments on embryonic metatarsal rudiments. As can be seen in Fig. 1, a and c, relatively low levels of collagenase-3 transcripts were found in cartilage from control samples. Labeling was restricted to some hypertrophic chondrocytes and cells localized in the perichondrium, but it was not detected in proliferating or in resting chondrocytes. By contrast, a high level of collagenase-3 expression was found in rudiments treated with 10−7 M RA for 7 d (Fig. 1, b and d). In these samples, expression of collagenase-3 was located mainly at an abnormally extended perichondrium, being especially marked in those zones in which it seems to invade into the underlying cartilage. In most cases, positive signal was found in spindle-shaped cells having oval nuclei and small size (Fig. 1 d). As in control animals, chondrocytes expressing collagenase-3 were low in number and restricted to the hypertrophic zone. The overexpression of collagenase-3 induced by RA was coupled to a series of morphological alterations in the metatarsal rudiments (Fig. 1, e and f). Thus, the longitudinal growth was reduced (control length, 3.1 ± 0.18 mm, n = 4; treatment with 10−8 RA, 2.7 ± 0.17 mm, n = 4, p < 0.05; treatment with 10−7 RA, 2.2 ± 0.14 mm, n = 4, p < 0.01). Histologically, the RA-treated metatarsal rudiments showed a decrease in the degree of structural anisotropy. As a result, boundaries between resting and proliferating zones appeared poorly defined and could hardly be recognized in these samples (Fig. 1 f). Likewise, RA treatment resulted in a partial inhibition of cellular enlargement during chondrocytic hypertrophy. In this way, hypertrophic-like chondrocytes showing mitotic figures were sometimes observed in RA-treated cartilages. Additionally, the perichondrium in RA-treated cultures appeared bigger and more irregular than in controls. Whereas the perichondrium in controls was only two to three layers thick (Fig. 1 e), the perichondrium in RA-treated cultures contained five to nine layers of cells, often invading into the underlying cartilage (Fig. 1 f). Proteoglycan content, as estimated by cytochemical staining with Alcian blue, was lower in RA-treated cartilages. This effect was especially evident in zones where the perichondrium expands and gives rise to cup-shaped depressions protruding into cartilage. An abrupt transition was evident between the weakly Alcian blue–stained intrachondral cells invading from the perichondrium and the strongly stained surrounding chondrocytes (Fig. 1 g). Such intrachondral cells were larger in size than surface perichondrial cells, presented basophilic cytoplasm (Fig. 1 h), and were histochemically positive for calcification (unpublished data). The characteristics of perichondrium-derived cells in RA-treated rudiments partially resembled those of osteoblasts and clearly differed from those of untreated bones where a sequential differentiation process from perichondrial cells to chondrocytes was observed.


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. Paraffin sections from embryonic metatarsal rudiments untreated (a and c) or treated with RA (b and d) were hybridized with a labeled antisense riboprobe for collagenase-3. In untreated cultures, collagenase-3 expression was relatively low, being restricted to a low number of cells located mainly in the perichondrium. (c) Higher magnification of a peripheric area showing that most collagenase-3–positive cells were small sized and flat shaped. Metatarsals treated with RA (b) showed higher levels of collagenase-3 expression. Positive signal was increased in zones in which the perichondrium invaded into the underlying cartilage (d). (e–h) Metatarsal sections untreated (e) and treated with RA (f–h) and stained with toluidine blue (e, f, and h) and Alcian blue (g). A clear boundary between proliferating and hypertrophic zones was observed in untreated metatarsals (e). RA-treated cultures showed a partial inhibition of cellular enlargement during chondrocytic hypertrophy resulting in ill-defined boundaries between proliferating and hypertrophic zones (f). Invasion of cartilage by cells from the perichondrium also increased in RA-treated cultures (f, arrowhead; g and h). Weakly Alcian blue–stained intrachondral cells invading from the perichondrium are observed in g. (h) Higher magnification of intrachondral cells showed in g and stained with toluidine blue. These cells are elongated and have basophilic cytoplasm. Bars: (a, e, and f) 100 μm; (b) 160 μm; (c, d, and g) 40 μm; (h) 25 μm.
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fig1: Collagenase-3 expression in metatarsal bone rudiments. Paraffin sections from embryonic metatarsal rudiments untreated (a and c) or treated with RA (b and d) were hybridized with a labeled antisense riboprobe for collagenase-3. In untreated cultures, collagenase-3 expression was relatively low, being restricted to a low number of cells located mainly in the perichondrium. (c) Higher magnification of a peripheric area showing that most collagenase-3–positive cells were small sized and flat shaped. Metatarsals treated with RA (b) showed higher levels of collagenase-3 expression. Positive signal was increased in zones in which the perichondrium invaded into the underlying cartilage (d). (e–h) Metatarsal sections untreated (e) and treated with RA (f–h) and stained with toluidine blue (e, f, and h) and Alcian blue (g). A clear boundary between proliferating and hypertrophic zones was observed in untreated metatarsals (e). RA-treated cultures showed a partial inhibition of cellular enlargement during chondrocytic hypertrophy resulting in ill-defined boundaries between proliferating and hypertrophic zones (f). Invasion of cartilage by cells from the perichondrium also increased in RA-treated cultures (f, arrowhead; g and h). Weakly Alcian blue–stained intrachondral cells invading from the perichondrium are observed in g. (h) Higher magnification of intrachondral cells showed in g and stained with toluidine blue. These cells are elongated and have basophilic cytoplasm. Bars: (a, e, and f) 100 μm; (b) 160 μm; (c, d, and g) 40 μm; (h) 25 μm.
Mentions: To study the putative effect of RA on the expression of collagenase-3 during bone formation, we first performed in situ hybridization experiments on embryonic metatarsal rudiments. As can be seen in Fig. 1, a and c, relatively low levels of collagenase-3 transcripts were found in cartilage from control samples. Labeling was restricted to some hypertrophic chondrocytes and cells localized in the perichondrium, but it was not detected in proliferating or in resting chondrocytes. By contrast, a high level of collagenase-3 expression was found in rudiments treated with 10−7 M RA for 7 d (Fig. 1, b and d). In these samples, expression of collagenase-3 was located mainly at an abnormally extended perichondrium, being especially marked in those zones in which it seems to invade into the underlying cartilage. In most cases, positive signal was found in spindle-shaped cells having oval nuclei and small size (Fig. 1 d). As in control animals, chondrocytes expressing collagenase-3 were low in number and restricted to the hypertrophic zone. The overexpression of collagenase-3 induced by RA was coupled to a series of morphological alterations in the metatarsal rudiments (Fig. 1, e and f). Thus, the longitudinal growth was reduced (control length, 3.1 ± 0.18 mm, n = 4; treatment with 10−8 RA, 2.7 ± 0.17 mm, n = 4, p < 0.05; treatment with 10−7 RA, 2.2 ± 0.14 mm, n = 4, p < 0.01). Histologically, the RA-treated metatarsal rudiments showed a decrease in the degree of structural anisotropy. As a result, boundaries between resting and proliferating zones appeared poorly defined and could hardly be recognized in these samples (Fig. 1 f). Likewise, RA treatment resulted in a partial inhibition of cellular enlargement during chondrocytic hypertrophy. In this way, hypertrophic-like chondrocytes showing mitotic figures were sometimes observed in RA-treated cartilages. Additionally, the perichondrium in RA-treated cultures appeared bigger and more irregular than in controls. Whereas the perichondrium in controls was only two to three layers thick (Fig. 1 e), the perichondrium in RA-treated cultures contained five to nine layers of cells, often invading into the underlying cartilage (Fig. 1 f). Proteoglycan content, as estimated by cytochemical staining with Alcian blue, was lower in RA-treated cartilages. This effect was especially evident in zones where the perichondrium expands and gives rise to cup-shaped depressions protruding into cartilage. An abrupt transition was evident between the weakly Alcian blue–stained intrachondral cells invading from the perichondrium and the strongly stained surrounding chondrocytes (Fig. 1 g). Such intrachondral cells were larger in size than surface perichondrial cells, presented basophilic cytoplasm (Fig. 1 h), and were histochemically positive for calcification (unpublished data). The characteristics of perichondrium-derived cells in RA-treated rudiments partially resembled those of osteoblasts and clearly differed from those of untreated bones where a sequential differentiation process from perichondrial cells to chondrocytes was observed.

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