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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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Osteocalcin and Cbfa1 expression in metatarsal bone rudiments treated with RA. Osteocalcin mRNA was found in RA-treated rudiments in a low number of cells located in zones in which perichondrium appeared extended into the underlying cartilage (a). Cells showing positive signal (arrowhead) were small sized and spindle shaped and partially overlapped those positive for collagenase-3. Osteocalcin expression was correlated with a decrease of proteoglycans as demonstrated by cytochemical detection with Alcian blue on paraffin sections (b, arrowhead). Cbfa1 expression was observed in both untreated rudiments (c) and treated with RA (d). In untreated cultures, Cbfa1 expression was low, being restricted to cells of the perichondrium (arrowhead) and some hypertrophic chondrocytes (asterisk). By contrast, metatarsals treated with RA (d) showed higher levels of Cbfa1 expression that partially overlapped that of collagenase-3. Positive signal was especially evident in central (diaphyseal) zones in which the perichondrium was expanded and typical chondrocytes were scarce (arrowhead). Bars: (a and b) 250 μm; (c and d) 67 μm.
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fig3: Osteocalcin and Cbfa1 expression in metatarsal bone rudiments treated with RA. Osteocalcin mRNA was found in RA-treated rudiments in a low number of cells located in zones in which perichondrium appeared extended into the underlying cartilage (a). Cells showing positive signal (arrowhead) were small sized and spindle shaped and partially overlapped those positive for collagenase-3. Osteocalcin expression was correlated with a decrease of proteoglycans as demonstrated by cytochemical detection with Alcian blue on paraffin sections (b, arrowhead). Cbfa1 expression was observed in both untreated rudiments (c) and treated with RA (d). In untreated cultures, Cbfa1 expression was low, being restricted to cells of the perichondrium (arrowhead) and some hypertrophic chondrocytes (asterisk). By contrast, metatarsals treated with RA (d) showed higher levels of Cbfa1 expression that partially overlapped that of collagenase-3. Positive signal was especially evident in central (diaphyseal) zones in which the perichondrium was expanded and typical chondrocytes were scarce (arrowhead). Bars: (a and b) 250 μm; (c and d) 67 μm.

Mentions: After treatment of cell cultures with RA, a clear time-dependent change in cell morphology was found. Both primary chondrocytes and RCS cells shifted from rounded polygonal shape to a very distinct flattened and more stellate shape, being such changes correlated with a decrease of proteoglycan content (unpublished data). In addition, an increase in calcium deposition was observed in RCS cells and primary cultures of chondrocytes. Previous studies have provided opposing results on the role that RA exerts on chondrocytic differentiation. Some works have shown that RA induces maturation and mineralization of chondrocytes (Iwamoto et al., 1994; Cancedda et al., 1995; Koyama et al., 1999), whereas other groups have reported that RA exerts an inhibitory effect on chondrocyte function (Ballock et al., 1994; De Luca et al., 2000). To analyze the molecular alterations associated with these morphological changes, we examined the putative occurrence of variations in the expression levels of different genes that could be associated with chondrocytic differentiation. Northern blot analysis revealed that type II collagen expression was strikingly downregulated after RA treatment of primary chondrocytes and RCS cells (Fig. 2, d and e). The loss of this cartilage-specific collagen suggested that chondrocytic cells had differentiated toward a mature hypertrophic chondrocyte or, alternatively, had dedifferentiated toward a fibroblastic phenotype. Hybridization of the blots with a probe for type X collagen showed the absence of detectable mRNA transcripts of this gene whose expression is characteristic of hypertrophic chondrocytes. However, hybridization of the same blots with probes for osteoblastic markers provided positive results. Thus, type I collagen positive signal was observed in RA-treated primary chondrocytes (Fig. 2 d). Similarly, osteocalcin mRNA was markedly induced in RA-treated RCS cells (Fig. 2 e), although the effect on primary cultures was virtually undetectable (unpublished data). These differences in the expression patterns of both types of RA-treated cells indicate that their response to retinoids is not identical, although they share several common RA-induced morphological and molecular alterations suggestive of their differentiation toward osteoblastic-like cells. In relation to this, it is remarkable the finding that matrix molecules, including osteocalcin, are only expressed in some populations of morphologically indistinguishable osteoblasts depending on variations in maturational status or in the microenvironment in which they are present (Candeliere et al., 2001). Consistent with this, in situ hybridization experiments on metatarsal rudiments revealed that osteocalcin transcripts were detected clearly in some cells from rudiments treated with 10−7 M RA for 7 d (Fig. 3 a) but not in control samples. In these RA-treated rudiments, expression of osteocalcin significantly overlapped that of collagenase-3, being found in a relatively low number of small spindle-shaped cells located in zones where the perichondrium appeared to protrude into the underlying cartilage (Fig. 3 a). Osteocalcin expression was negatively correlated with proteoglycan content (Fig. 3 b).


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)

Osteocalcin and Cbfa1 expression in metatarsal bone rudiments treated with RA. Osteocalcin mRNA was found in RA-treated rudiments in a low number of cells located in zones in which perichondrium appeared extended into the underlying cartilage (a). Cells showing positive signal (arrowhead) were small sized and spindle shaped and partially overlapped those positive for collagenase-3. Osteocalcin expression was correlated with a decrease of proteoglycans as demonstrated by cytochemical detection with Alcian blue on paraffin sections (b, arrowhead). Cbfa1 expression was observed in both untreated rudiments (c) and treated with RA (d). In untreated cultures, Cbfa1 expression was low, being restricted to cells of the perichondrium (arrowhead) and some hypertrophic chondrocytes (asterisk). By contrast, metatarsals treated with RA (d) showed higher levels of Cbfa1 expression that partially overlapped that of collagenase-3. Positive signal was especially evident in central (diaphyseal) zones in which the perichondrium was expanded and typical chondrocytes were scarce (arrowhead). Bars: (a and b) 250 μm; (c and d) 67 μm.
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fig3: Osteocalcin and Cbfa1 expression in metatarsal bone rudiments treated with RA. Osteocalcin mRNA was found in RA-treated rudiments in a low number of cells located in zones in which perichondrium appeared extended into the underlying cartilage (a). Cells showing positive signal (arrowhead) were small sized and spindle shaped and partially overlapped those positive for collagenase-3. Osteocalcin expression was correlated with a decrease of proteoglycans as demonstrated by cytochemical detection with Alcian blue on paraffin sections (b, arrowhead). Cbfa1 expression was observed in both untreated rudiments (c) and treated with RA (d). In untreated cultures, Cbfa1 expression was low, being restricted to cells of the perichondrium (arrowhead) and some hypertrophic chondrocytes (asterisk). By contrast, metatarsals treated with RA (d) showed higher levels of Cbfa1 expression that partially overlapped that of collagenase-3. Positive signal was especially evident in central (diaphyseal) zones in which the perichondrium was expanded and typical chondrocytes were scarce (arrowhead). Bars: (a and b) 250 μm; (c and d) 67 μm.
Mentions: After treatment of cell cultures with RA, a clear time-dependent change in cell morphology was found. Both primary chondrocytes and RCS cells shifted from rounded polygonal shape to a very distinct flattened and more stellate shape, being such changes correlated with a decrease of proteoglycan content (unpublished data). In addition, an increase in calcium deposition was observed in RCS cells and primary cultures of chondrocytes. Previous studies have provided opposing results on the role that RA exerts on chondrocytic differentiation. Some works have shown that RA induces maturation and mineralization of chondrocytes (Iwamoto et al., 1994; Cancedda et al., 1995; Koyama et al., 1999), whereas other groups have reported that RA exerts an inhibitory effect on chondrocyte function (Ballock et al., 1994; De Luca et al., 2000). To analyze the molecular alterations associated with these morphological changes, we examined the putative occurrence of variations in the expression levels of different genes that could be associated with chondrocytic differentiation. Northern blot analysis revealed that type II collagen expression was strikingly downregulated after RA treatment of primary chondrocytes and RCS cells (Fig. 2, d and e). The loss of this cartilage-specific collagen suggested that chondrocytic cells had differentiated toward a mature hypertrophic chondrocyte or, alternatively, had dedifferentiated toward a fibroblastic phenotype. Hybridization of the blots with a probe for type X collagen showed the absence of detectable mRNA transcripts of this gene whose expression is characteristic of hypertrophic chondrocytes. However, hybridization of the same blots with probes for osteoblastic markers provided positive results. Thus, type I collagen positive signal was observed in RA-treated primary chondrocytes (Fig. 2 d). Similarly, osteocalcin mRNA was markedly induced in RA-treated RCS cells (Fig. 2 e), although the effect on primary cultures was virtually undetectable (unpublished data). These differences in the expression patterns of both types of RA-treated cells indicate that their response to retinoids is not identical, although they share several common RA-induced morphological and molecular alterations suggestive of their differentiation toward osteoblastic-like cells. In relation to this, it is remarkable the finding that matrix molecules, including osteocalcin, are only expressed in some populations of morphologically indistinguishable osteoblasts depending on variations in maturational status or in the microenvironment in which they are present (Candeliere et al., 2001). Consistent with this, in situ hybridization experiments on metatarsal rudiments revealed that osteocalcin transcripts were detected clearly in some cells from rudiments treated with 10−7 M RA for 7 d (Fig. 3 a) but not in control samples. In these RA-treated rudiments, expression of osteocalcin significantly overlapped that of collagenase-3, being found in a relatively low number of small spindle-shaped cells located in zones where the perichondrium appeared to protrude into the underlying cartilage (Fig. 3 a). Osteocalcin expression was negatively correlated with proteoglycan content (Fig. 3 b).

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