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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 RA on expression of collagenase-3 and different chondrocytic and osteoblastic markers. (a) Primary chondrocytes were exposed to 10−6 M RA for the times shown, and then total RNA was extracted and analyzed by Northern blot with collagenase-3 and MT1-MMP cDNA probes. 28S rRNA stained with ethidium bromide is shown as loading control. (b) RCS cells were incubated with 10−6 M RA for the times shown, and total RNA was then analyzed as in a. (c) RCS cells were treated with 10−6 RA for the times indicated, and proteins in conditioned media were analyzed by Western blot using an antibody against collagenase-3. Cells were also treated for 72 h with different RA concentrations, and collagenase-3 protein secreted to media was detected. Primary chondrocytes (d) or RCS cells (e) were induced with 10−6 M RA for the times shown, and total RNA was analyzed by Northern blot with specific probes for the indicated genes.
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fig2: Effect of RA on expression of collagenase-3 and different chondrocytic and osteoblastic markers. (a) Primary chondrocytes were exposed to 10−6 M RA for the times shown, and then total RNA was extracted and analyzed by Northern blot with collagenase-3 and MT1-MMP cDNA probes. 28S rRNA stained with ethidium bromide is shown as loading control. (b) RCS cells were incubated with 10−6 M RA for the times shown, and total RNA was then analyzed as in a. (c) RCS cells were treated with 10−6 RA for the times indicated, and proteins in conditioned media were analyzed by Western blot using an antibody against collagenase-3. Cells were also treated for 72 h with different RA concentrations, and collagenase-3 protein secreted to media was detected. Primary chondrocytes (d) or RCS cells (e) were induced with 10−6 M RA for the times shown, and total RNA was analyzed by Northern blot with specific probes for the indicated genes.

Mentions: To further study the effect of RA on the expression of collagenase-3, we first used primary chondrocyte cultures. Cells were treated with 10−6 M RA, and total RNA was obtained at different times and analyzed by Northern blot using a specific collagenase-3 probe. As shown in Fig. 2 a, RA induced the accumulation of a 2.9-kb mRNA transcript corresponding to collagenase-3, the maximal effect being reached between 24 and 48 h and declining at longer times of incubation. Similar results were obtained when rat chondrosarcoma (RCS) cells were used as the experimental model. The main advantage of using this cell line derives from the fact that its chondrocyte phenotype is extremely stable in standard tissue culture conditions compared with the unstable phenotype exhibited by other cells from the same lineage (Mukhopadhyay et al., 1995). RCS cells treated with 10−6 M RA also showed a marked induction of collagenase-3 expression (Fig. 2 b). In addition, a dose–response analysis showed that as little as 10−7 M RA induced a detectable expression of collagenase-3 mRNA, whereas incubation of the cells with 10−5 M induced a maximal accumulation of this mRNA (unpublished data). To determine if the inducing effect of RA on collagenase-3 mRNA levels was also reflected at the protein level, we performed Western blot analysis with conditioned medium from RCS cells treated with RA. As can be seen in Fig. 2 c, a band immunoreactive against collagenase-3 monoclonal antibodies was detected in medium from cells treated with 10−6 M RA for 48 h. This band was absent in medium obtained from control untreated cells. A time course analysis demonstrated that the maximum level of collagenase-3 protein was detected in cells treated with RA for 72 h (Fig. 2 c).


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 RA on expression of collagenase-3 and different chondrocytic and osteoblastic markers. (a) Primary chondrocytes were exposed to 10−6 M RA for the times shown, and then total RNA was extracted and analyzed by Northern blot with collagenase-3 and MT1-MMP cDNA probes. 28S rRNA stained with ethidium bromide is shown as loading control. (b) RCS cells were incubated with 10−6 M RA for the times shown, and total RNA was then analyzed as in a. (c) RCS cells were treated with 10−6 RA for the times indicated, and proteins in conditioned media were analyzed by Western blot using an antibody against collagenase-3. Cells were also treated for 72 h with different RA concentrations, and collagenase-3 protein secreted to media was detected. Primary chondrocytes (d) or RCS cells (e) were induced with 10−6 M RA for the times shown, and total RNA was analyzed by Northern blot with specific probes for the indicated genes.
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Related In: Results  -  Collection

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fig2: Effect of RA on expression of collagenase-3 and different chondrocytic and osteoblastic markers. (a) Primary chondrocytes were exposed to 10−6 M RA for the times shown, and then total RNA was extracted and analyzed by Northern blot with collagenase-3 and MT1-MMP cDNA probes. 28S rRNA stained with ethidium bromide is shown as loading control. (b) RCS cells were incubated with 10−6 M RA for the times shown, and total RNA was then analyzed as in a. (c) RCS cells were treated with 10−6 RA for the times indicated, and proteins in conditioned media were analyzed by Western blot using an antibody against collagenase-3. Cells were also treated for 72 h with different RA concentrations, and collagenase-3 protein secreted to media was detected. Primary chondrocytes (d) or RCS cells (e) were induced with 10−6 M RA for the times shown, and total RNA was analyzed by Northern blot with specific probes for the indicated genes.
Mentions: To further study the effect of RA on the expression of collagenase-3, we first used primary chondrocyte cultures. Cells were treated with 10−6 M RA, and total RNA was obtained at different times and analyzed by Northern blot using a specific collagenase-3 probe. As shown in Fig. 2 a, RA induced the accumulation of a 2.9-kb mRNA transcript corresponding to collagenase-3, the maximal effect being reached between 24 and 48 h and declining at longer times of incubation. Similar results were obtained when rat chondrosarcoma (RCS) cells were used as the experimental model. The main advantage of using this cell line derives from the fact that its chondrocyte phenotype is extremely stable in standard tissue culture conditions compared with the unstable phenotype exhibited by other cells from the same lineage (Mukhopadhyay et al., 1995). RCS cells treated with 10−6 M RA also showed a marked induction of collagenase-3 expression (Fig. 2 b). In addition, a dose–response analysis showed that as little as 10−7 M RA induced a detectable expression of collagenase-3 mRNA, whereas incubation of the cells with 10−5 M induced a maximal accumulation of this mRNA (unpublished data). To determine if the inducing effect of RA on collagenase-3 mRNA levels was also reflected at the protein level, we performed Western blot analysis with conditioned medium from RCS cells treated with RA. As can be seen in Fig. 2 c, a band immunoreactive against collagenase-3 monoclonal antibodies was detected in medium from cells treated with 10−6 M RA for 48 h. This band was absent in medium obtained from control untreated cells. A time course analysis demonstrated that the maximum level of collagenase-3 protein was detected in cells treated with RA for 72 h (Fig. 2 c).

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