Transcription from the tartrate-resistant acid phosphatase promoter is negatively regulated by the Myc oncoprotein.

TRAP, a characteristic marker of osteoclast differentiation, is an enzyme that plays an active role in the process of bone resorption. Despite the importance of TRAP in osteoclast biology, the components involved in the transcriptional regulation of this gene are largely unknown. This study investigated the regulation of TRAP transcription by the Myc oncoprotein in three different cell types. A series of nested TRAP promoter deletion constructs were cotransfected into P388D1 murine macrophages and C3H10T1/2 murine embryonic fibroblasts along with a backbone plasmid control or expression plasmids containing v-Myc, c-Myc, or an inactive v-Myc protein construct (delta84/NLS). Both v-Myc and c-Myc negatively regulated transcription from the TRAP promoter in P388D1 and C3H10T1/2 cells, 90% and 50%, respective to cell type and amount of endogenous Myc protein, and delta84/NLS had no effect. The functional Myc-responsive element(s) within the TRAP promoter was localized to a region between -436 and +1 bp, which contains two putative Myc-inhibitory binding sites coincident with an initiator element (Inr) at -116 bp and -18 bp. Conversely, in the HD-11EM chicken v-Myc transformed preosteoclast cell line, the full-length TRAP promoter transcription was increased when endogenous v-Myc levels were decreased in response to pretreatment of these cells with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This report provides the first evidence of the specific regulation of TRAP at the transcriptional level by Myc, a transcription factor that is normally expressed at relatively high levels in preosteoclasts and other myelomonocytic cells and suggests that Myc plays an active role in suppressing the transcription of a mature osteoclast selective gene.

Long-term in vitro analysis of limb cartilage development: involvement of Wnt signaling.

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.

Frizzled-7 and limb mesenchymal chondrogenesis: effect of misexpression and involvement of N-cadherin.

Products of the Frizzled family of tissue polarity genes have been identified as putative receptors for the Wnt family of signaling molecules. Wnt-signaling is implicated in the regulation of limb mesenchymal chondrogenesis, and our recent study indicates that N-cadherin and related activities are functionally involved in Wnt-7a-mediated inhibition of chondrogenesis. By using an in vitro high-density micromass culture system of chick limb mesenchymal cells, we have analyzed the spatiotemporal expression patterns and the effects on chondrogenesis of RCAS retroviral-mediated misexpression of Chfz-1 and Chfz-7, two Frizzled genes implicated in chondrogenic regulation. Chfz-1 expression was localized at areas surrounding the cartilaginous nodules at all time points examined, whereas Chfz-7 expression was limited to cellular aggregates during initial mesenchymal condensation, and subsequently was down-regulated from the centers toward the periphery of cartilage nodules at the time of chondrogenic differentiation, resembling the pattern of N-cadherin expression. Chondrogenesis in vitro was inhibited and limited to a smaller area of the culture upon misexpression of Chfz-7, but not affected by Chfz-1 misexpression. Analyses of cellular condensation and chondrogenic differentiation showed that the inhibitory action of Chfz-7 is unlikely to be at the chondrogenic differentiation step, but instead affects the earlier precartilage aggregate formation event. At 24 hr, expression of N-cadherin, a key component of the cellular condensation phase of chondrogenesis, was delayed/suppressed in Chfz-7 misexpressing cultures, and was limited to a significantly smaller cellular condensation area within the entire culture at 48 hr, when compared with control cultures. Chfz-1 misexpressing cultures appeared similar to control cultures at all time points. However, neither Chfz-1 nor Chfz-7 misexpression affected mesenchymal cell proliferation in vitro. These results suggest that Chfz-7 is active in regulating N-cadherin expression during the process of limb mesenchymal chondrogenesis and that Chfz-1 and Chfz-7 are involved in different Wnt-signaling pathways.

AP-1 transcription factor complex is a target of signals from both WnT-7a and N-cadherin-dependent cell-cell adhesion complex during the regulation of limb mesenchymal chondrogenesis.

Wnt signaling has been implicated in the regulation of limb mesenchymal chondrogenesis. In this study, we have analyzed the molecular mechanism of Wnt-7a inhibition of chondrogenic differentiation by examining the involvement of mitogen-activated protein kinase (MAPK) pathways, i.e., Erk and p38. The combination of Wnt-7a misexpression and Erk inhibition partially recovers Wnt-7a inhibition of chondrogenic differentiation, whereas the combination of Wnt-7a misexpression and p38 inhibition acts in a synergistic chondro-inhibitory fashion. Although Wnt-7a misexpression has no direct effect on Erk signaling, it increases the activity of one of the ultimate targets of the MAPK pathway, c-jun, a major component of the activator protein-1 (AP-1) transcription factor complex. In addition, Wnt-7a misexpression enhances the activity of an AP-1 promoter-luciferase reporter construct by approximately 2.3-fold in vitro. Interestingly, misexpression of wild-type N-cadherin in these micromass cultures suppresses the activity of the same AP-1 promoter by approximately 40%, whereas misexpression of an extracellular 390-amino-acid N-terminal deletion mutant of N-cadherin has a stimulatory effect on the AP-1 promoter activity by approximately 2.6-fold. Thus, our results suggest that at least a part of the chondro-inhibitory effect of Wnt-7a misexpression may involve AP-1 transcription factor stimulation. Furthermore, a very tightly regulated level of AP-1 activity is necessary for the process of limb mesenchymal chondrogenesis, and signals from Wnt-ligands (e.g., Wnt-7a), cell adhesion molecules (e.g., N-cadherin), and MAPK pathways (e.g., Erk and p38) are interactively involved in this regulation.