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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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Implication of p38 MAPK in collagenase-3 induction by RA. (a) RCS cells were treated with 10−6 M RA for the times indicated. Cells were lysed, and protein extracts were analyzed by Western blot. The levels of activated ERK1/2, JNK1/2, and p38 were determined using phosphospecific antibodies for the corresponding MAPKs (p-ERK1/2, p-JNK1/2, p-p38). As controls, levels of total ERK1/2 and p38 were determined with specific antibodies. Cell lysates from HaCaT cells treated for 20 min with 10−7 M TPA or 20 ng/ml TNF-α were used as positive controls for activated ERK1/2 or JNK1/2 and p38, respectively. (b) Increasing amounts of p38 inhibitor SB 203580 or ERK1/2 inhibitor PD 98059 (μg/μl) were added to the culture medium 1 h before inducing RCS cells with 10−6 M RA. Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot. The same filter was subsequently hybridized with probes specific for Cbfa1 and osteocalcin.
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fig8: Implication of p38 MAPK in collagenase-3 induction by RA. (a) RCS cells were treated with 10−6 M RA for the times indicated. Cells were lysed, and protein extracts were analyzed by Western blot. The levels of activated ERK1/2, JNK1/2, and p38 were determined using phosphospecific antibodies for the corresponding MAPKs (p-ERK1/2, p-JNK1/2, p-p38). As controls, levels of total ERK1/2 and p38 were determined with specific antibodies. Cell lysates from HaCaT cells treated for 20 min with 10−7 M TPA or 20 ng/ml TNF-α were used as positive controls for activated ERK1/2 or JNK1/2 and p38, respectively. (b) Increasing amounts of p38 inhibitor SB 203580 or ERK1/2 inhibitor PD 98059 (μg/μl) were added to the culture medium 1 h before inducing RCS cells with 10−6 M RA. Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot. The same filter was subsequently hybridized with probes specific for Cbfa1 and osteocalcin.

Mentions: On the other hand, it is well established that activation of tyrosine kinase-dependent signaling can activate downstream signaling cascades including MAPKs. To elucidate the putative implication of extracellular signal–regulated kinase (ERK)1,2, Jun NH2-terminal kinase (JNK)1, and p38 MAPKs pathways in mediating the RA-dependent induction of collagenase-3 expression in RCS cells, we studied the activation of these different kinases after RA treatment. As illustrated in Fig. 8 a, only p38 showed constitutive levels of activation and was appreciably activated after a 90-min RA treatment. This effect continued for at least 12 h. To test the implication of p38 in RA-elicited collagenase-3 induction, we pretreated RCS cells with the p38 inhibitor SB 203580 and then analyzed the obtained RNA after incubation with RA. As shown in Fig. 8 b, treatment of these cells with SB 203580 abolished the RA-induced expression of collagenase-3. In contrast, the ERK1,2 pathway inhibitor PD 98059 strongly augmented collagenase-3 induction by RA in RCS cells. It is remarkable that PD 98059 alone, in the absence of RA, did not elicit any inductive effect on collagenase-3 expression. We also examined if the actions of these MAPK inhibitors could be extended to other RA-induced genes such as Cbfa1 and osteocalcin. As shown in Fig. 8 b, the p38 inhibitor SB 203580 abolished the RA-induced expression of Cbfa1 and osteocalcin, whereas PD 98059 increased the RA-mediated induction of both genes. These results provide evidence that the p38 MAPK signaling pathway seems essential for expression of the different genes induced by RA in chondrocytic cells, whereas ERK1,2 MAPKs play an inhibitory role on the process.


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)

Implication of p38 MAPK in collagenase-3 induction by RA. (a) RCS cells were treated with 10−6 M RA for the times indicated. Cells were lysed, and protein extracts were analyzed by Western blot. The levels of activated ERK1/2, JNK1/2, and p38 were determined using phosphospecific antibodies for the corresponding MAPKs (p-ERK1/2, p-JNK1/2, p-p38). As controls, levels of total ERK1/2 and p38 were determined with specific antibodies. Cell lysates from HaCaT cells treated for 20 min with 10−7 M TPA or 20 ng/ml TNF-α were used as positive controls for activated ERK1/2 or JNK1/2 and p38, respectively. (b) Increasing amounts of p38 inhibitor SB 203580 or ERK1/2 inhibitor PD 98059 (μg/μl) were added to the culture medium 1 h before inducing RCS cells with 10−6 M RA. Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot. The same filter was subsequently hybridized with probes specific for Cbfa1 and osteocalcin.
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Related In: Results  -  Collection

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fig8: Implication of p38 MAPK in collagenase-3 induction by RA. (a) RCS cells were treated with 10−6 M RA for the times indicated. Cells were lysed, and protein extracts were analyzed by Western blot. The levels of activated ERK1/2, JNK1/2, and p38 were determined using phosphospecific antibodies for the corresponding MAPKs (p-ERK1/2, p-JNK1/2, p-p38). As controls, levels of total ERK1/2 and p38 were determined with specific antibodies. Cell lysates from HaCaT cells treated for 20 min with 10−7 M TPA or 20 ng/ml TNF-α were used as positive controls for activated ERK1/2 or JNK1/2 and p38, respectively. (b) Increasing amounts of p38 inhibitor SB 203580 or ERK1/2 inhibitor PD 98059 (μg/μl) were added to the culture medium 1 h before inducing RCS cells with 10−6 M RA. Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot. The same filter was subsequently hybridized with probes specific for Cbfa1 and osteocalcin.
Mentions: On the other hand, it is well established that activation of tyrosine kinase-dependent signaling can activate downstream signaling cascades including MAPKs. To elucidate the putative implication of extracellular signal–regulated kinase (ERK)1,2, Jun NH2-terminal kinase (JNK)1, and p38 MAPKs pathways in mediating the RA-dependent induction of collagenase-3 expression in RCS cells, we studied the activation of these different kinases after RA treatment. As illustrated in Fig. 8 a, only p38 showed constitutive levels of activation and was appreciably activated after a 90-min RA treatment. This effect continued for at least 12 h. To test the implication of p38 in RA-elicited collagenase-3 induction, we pretreated RCS cells with the p38 inhibitor SB 203580 and then analyzed the obtained RNA after incubation with RA. As shown in Fig. 8 b, treatment of these cells with SB 203580 abolished the RA-induced expression of collagenase-3. In contrast, the ERK1,2 pathway inhibitor PD 98059 strongly augmented collagenase-3 induction by RA in RCS cells. It is remarkable that PD 98059 alone, in the absence of RA, did not elicit any inductive effect on collagenase-3 expression. We also examined if the actions of these MAPK inhibitors could be extended to other RA-induced genes such as Cbfa1 and osteocalcin. As shown in Fig. 8 b, the p38 inhibitor SB 203580 abolished the RA-induced expression of Cbfa1 and osteocalcin, whereas PD 98059 increased the RA-mediated induction of both genes. These results provide evidence that the p38 MAPK signaling pathway seems essential for expression of the different genes induced by RA in chondrocytic cells, whereas ERK1,2 MAPKs play an inhibitory role on the process.

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