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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

Collagenase-3 expression in metatarsal bone rudiments from embryos with targeted deletion of the Cbfa1 and MT1-MMP genes. Cbfa1-null (a–d) and MT1-MMP–null (e–h) metatarsal rudiments were untreated (a, b, e, and f) or treated with RA (c, d, g, and h) and stained with Alcian blue and nuclear fast red (a, c, e, and g) or hybridized with a labeled antisense riboprobe for collagenase-3 (b, d, f, and h). Untreated Cbfa1^−/− rudiments showed signs of cellular disorganization, especially in the hypertrophic zone where a clear Alcian blue staining was observed (a). Treatment of Cbfa1^−/− metatarsi with RA resulted in a marked inhibition of the chondrocytic hypertrophy (c). In situ hybridization studies failed to find positive signal for collagenase-3 in either untreated (b) or RA-treated (d) rudiments. Untreated MT1-MMP^−/− rudiments showed a disorganized hypertrophic zone with an intense Alcian blue staining (e). Collagenase-3 expression was low but could be observed in cells located mainly in the perichondrium (f). Treatment of MT1-MMP^−/− metatarsi with RA induced some cytological changes (g) and resulted in an increased collagenase-3 expression (h) at the hypertrophic chondrocytes. Bars, 200 μm.

Figure 5.

Expression of RA receptors in RCS cells. Cells were induced with 10⁻⁶ M RA for the times indicated, and total RNA was analyzed by Northern blot with specific probes for αRAR and γRAR.

Figure 6.

Effect of different RA receptors agonists and antagonists on collagenase-3 expression. RCS cells were cultured for 48 h in the presence of 10⁻⁶ M of different agonists (Ro40-6055, αRAR agonist; Ro48-2249, βRAR agonist; Ro44-4753, γRAR agonist; RXR agonist, LG100064) (a) or with 10⁻⁶ M RA plus different antagonists (Ro41-5253, αRAR antagonist; LE135, βRAR antagonist) (b). Total RNA was extracted, and collagenase-3 transcripts were detected by Northern blot.

Figure 7.

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.

Figure 8.

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.