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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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Analysis of the signaling pathways involved in RA-mediated induction of collagenase-3 expression. (a) Effect of cycloheximide (CHX) (0.5 μg/ml) on collagenase-3 mRNA levels induced in RCS cells treated with 10−6 M RA for 48 h. (b) Effect of inhibitors of different intracellular signaling pathways on RA-induced collagenase-3 mRNA levels in RCS cells. Genistein (100 μg/ml), staurosporine (2 nM), GF109203X (5 μM), H89 (500 nM), and indomethacin (5 μM) were added to the culture medium 1 h before induction with 10−6 M RA. Total RNA was extracted after 48 h and analyzed by Northern blot with a collagenase-3 probe.
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fig7: Analysis of the signaling pathways involved in RA-mediated induction of collagenase-3 expression. (a) Effect of cycloheximide (CHX) (0.5 μg/ml) on collagenase-3 mRNA levels induced in RCS cells treated with 10−6 M RA for 48 h. (b) Effect of inhibitors of different intracellular signaling pathways on RA-induced collagenase-3 mRNA levels in RCS cells. Genistein (100 μg/ml), staurosporine (2 nM), GF109203X (5 μM), H89 (500 nM), and indomethacin (5 μM) were added to the culture medium 1 h before induction with 10−6 M RA. Total RNA was extracted after 48 h and analyzed by Northern blot with a collagenase-3 probe.

Mentions: To provide further insights into the mechanisms underlying the upregulating effect of RA on collagenase-3 expression in chondrocytic cells, we performed cell culture experiments in the presence of the protein synthesis inhibitor cycloheximide. As shown in Fig. 7 a, incubation of RCS cells with cycloheximide blocked the effect of RA on collagenase-3 mRNA levels. Therefore, we conclude that de novo protein synthesis is required for collagenase-3 induction by RA. We next evaluated the possibility that different signaling pathways could be involved in this process. To this purpose, RCS cells were first incubated with RA in the presence or absence of several inhibitors of these signaling pathways, and the levels of collagenase-3 were examined by Northern blot. As illustrated in Fig. 7 b, the highly specific PKC inhibitor GF109203X diminished the upregulating effect of RA on collagenase-3 expression, indicating the involvement of a PKC in this process. However, the classical but less specific PKC inhibitor staurosporine not only was unable to diminish its expression but even enhanced it, likely through an alternative mechanism (Shoshan and Linder, 1994). To determine whether a tyrosine kinase was also involved in collagenase-3 induction, RCS cells were incubated with RA in the presence or absence of genistein. As shown in Fig. 7 b, this inhibitor blocked the induction of collagenase-3 expression elicited by RA. By contrast, incubation of RCS cells with H89, a protein kinase A inhibitor, or with indomethacin, which blocks prostaglandin synthesis, did not diminish RA-mediated induction of collagenase-3 and even promoted it (Fig. 7 b). Taken together, these results indicate that the positive effect of RA on collagenase-3 expression in RCS cells is exerted through a signaling pathway involving PKC and tyrosine kinase activities.


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)

Analysis of the signaling pathways involved in RA-mediated induction of collagenase-3 expression. (a) Effect of cycloheximide (CHX) (0.5 μg/ml) on collagenase-3 mRNA levels induced in RCS cells treated with 10−6 M RA for 48 h. (b) Effect of inhibitors of different intracellular signaling pathways on RA-induced collagenase-3 mRNA levels in RCS cells. Genistein (100 μg/ml), staurosporine (2 nM), GF109203X (5 μM), H89 (500 nM), and indomethacin (5 μM) were added to the culture medium 1 h before induction with 10−6 M RA. Total RNA was extracted after 48 h and analyzed by Northern blot with a collagenase-3 probe.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Analysis of the signaling pathways involved in RA-mediated induction of collagenase-3 expression. (a) Effect of cycloheximide (CHX) (0.5 μg/ml) on collagenase-3 mRNA levels induced in RCS cells treated with 10−6 M RA for 48 h. (b) Effect of inhibitors of different intracellular signaling pathways on RA-induced collagenase-3 mRNA levels in RCS cells. Genistein (100 μg/ml), staurosporine (2 nM), GF109203X (5 μM), H89 (500 nM), and indomethacin (5 μM) were added to the culture medium 1 h before induction with 10−6 M RA. Total RNA was extracted after 48 h and analyzed by Northern blot with a collagenase-3 probe.
Mentions: To provide further insights into the mechanisms underlying the upregulating effect of RA on collagenase-3 expression in chondrocytic cells, we performed cell culture experiments in the presence of the protein synthesis inhibitor cycloheximide. As shown in Fig. 7 a, incubation of RCS cells with cycloheximide blocked the effect of RA on collagenase-3 mRNA levels. Therefore, we conclude that de novo protein synthesis is required for collagenase-3 induction by RA. We next evaluated the possibility that different signaling pathways could be involved in this process. To this purpose, RCS cells were first incubated with RA in the presence or absence of several inhibitors of these signaling pathways, and the levels of collagenase-3 were examined by Northern blot. As illustrated in Fig. 7 b, the highly specific PKC inhibitor GF109203X diminished the upregulating effect of RA on collagenase-3 expression, indicating the involvement of a PKC in this process. However, the classical but less specific PKC inhibitor staurosporine not only was unable to diminish its expression but even enhanced it, likely through an alternative mechanism (Shoshan and Linder, 1994). To determine whether a tyrosine kinase was also involved in collagenase-3 induction, RCS cells were incubated with RA in the presence or absence of genistein. As shown in Fig. 7 b, this inhibitor blocked the induction of collagenase-3 expression elicited by RA. By contrast, incubation of RCS cells with H89, a protein kinase A inhibitor, or with indomethacin, which blocks prostaglandin synthesis, did not diminish RA-mediated induction of collagenase-3 and even promoted it (Fig. 7 b). Taken together, these results indicate that the positive effect of RA on collagenase-3 expression in RCS cells is exerted through a signaling pathway involving PKC and tyrosine kinase activities.

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