Periodontal ligament (PDL) cells are known to play important roles in tooth eruption and alveolar bone metabolism. We previously reported that PTHrP increases RANKL expression in human PDL cells, suggesting that it promotes odontoclastic root resorption during tooth eruption. While it is known that Notch-related genes play a key role during bone development, the role of the Notch signaling pathway in PDL cells during tooth and bone resorption is less clear. We hypothesized that PTHrP induces a Notch ligand in PDL cells and thereby regulates osteo- and odontoclastogenesis. We found that PTHrP increased Notch1 ligand Jagged1 expression in human PDL cells in a dose- and time-dependent manner. PTHrP-induced Jagged1 up-regulation was mediated by PKA activation, but not by PKC. Jagged1 also promoted RANKL-induced osteoclastogenesis. These results demonstrate that PTHrP induces Jagged1 expression in PDL cells, leading to osteo- and odontoclastogenesis, and thus likely promoting tooth and alveolar bone resorption.

No abstract

Recent studies using SOCS family knock-out mice have suggested that SOCS proteins have multiple biological functions in addition to their role as negative regulators of JAK-STAT signaling. To explore these other functions of this family of proteins, we used yeast two-hybrid screening to find proteins interacting with human SOCS-3. We identified the transcriptional factor DP-1 as a SOCS-3-interacting protein involved in regulation of the cell cycle. Immunoprecipitation-Western blot assay showed that this interaction between these endogenous proteins occurred in cells both in vitro and in vivo. SOCS-3 interacted with the C-terminal region of DP-1, and amino acids 156 -172 of SOCS-3 were required for this interaction. Confocal microscopy revealed that SOCS-3 and DP-1 were primarily colocalized in the cytoplasm. SOCS-3 inhibited E2F/DP-1 transcriptional activity under the cyclin-E promoter and actually inhibited cell cycle progression and cell growth under E2F/DP-1 control. In contrast, DP-1 almost completely eliminated the inhibitory action of SOCS-3 on LIF-stimulated STAT-3 transcriptional activity in JAK-STAT signaling. Interestingly, the alternative regulatory action of SOCS-3 and DP-1 was dramatically eliminated by each siRNA. Taken together, these findings demonstrate that SOCS-3 acts as a negative regulator of the cell cycle progression under E2F/DP-1 control by interfering with heterodimer formation between DP-1 and E2F and also that DP-1 plays an important role in controlling JAK-STAT signaling.Members of the family of suppressor of cytokine signaling (SOCS), 2 designated SOCS-1 to SOCS-7 and CIS, are induced by stimulation via several kinds of cytokines and growth factors (1-3). These proteins regulate JAK-STAT signaling in a classical negative feedback loop of the signaling cascade (4, 5). SOCS family proteins also act as an important regulator of cell differentiation, as evidenced by the following findings: SOCS-1 suppresses muscle differentiation (6); SOCS-2 regulates neuronal differentiation (7); SOCS-3 induces myoblast differentiation (8); and SOCS-3 and SOCS-5 are involved in T helper cell differentiation (9, 10). In addition, these proteins are thought to strongly contribute to the development and progression of several kinds of tumors such as hepatocellular carcinoma (11-13), chronic myeloid leukemia (14), ovarian and breast carcinoma (15), and so on (16 -21). These observations suggested to us that SOCS family proteins might exhibit multipotential functions as regulators of cell differentiation and tumor cell growth besides being a negative regulator of JAK-STAT signaling. To explore this possibility, we considered that identification of SOCS-interacting proteins would be an extraordinarily good strategy. Thus, using the yeast two-hybrid screening system, we sought to identify presently proteins interacting with human SOCS-3. As a result, we found DP-1, a transcriptional factor for cell cycle regulation, to be such a SOCS-3-interacting protein.DP-1 was first identified as a partner protein of E2F-...

The cell cycle-regulating transcription factors DP-1 and E2F form a heterodimeric complex and play a central role in cell cycle progression. Two different DP subunits (DP-1 and DP-2) exist in humans. In this study, we identified two novel DP-1 isoforms (DP-1␣ and DP-1␤) and characterized their structure and function. DP-1␣ is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1␤ is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast twohybrid and immunoprecipitation assays demonstrated that DP-1␣ binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1␤. Immunofluorescence analysis revealed that the subcellular localization of both DP-1 isoforms changed from the cytoplasm to the nucleus in HEK 293 cells cotransfected with E2F1 and wild-type DP-1 or DP-1␤. However, such a translocation for DP-1␣ was barely observed. Reverse transcription-PCR results showed that the three DP-1 isoforms are expressed ubiquitously at equal levels in several normal human tissues. We also demonstrated the expression of these isoforms at the protein level by Western blotting. Interestingly, we observed a significant decrease in transcriptional activity, a marked delay of cell cycle progression, and an inhibition of cell proliferation in DP-1␣-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1␣ is a novel isoform of DP-1 that acts as a dominantnegative regulator of cell cycle progression.The E2F family of transcription factors plays an essential role in regulating cell cycle progression (1, 2). This family consists of two subgroups, termed E2F and DP. Currently, the E2F/DP family in mammals is known to include seven E2F members (E2F1-7) and two DP members (DP-1 and DP-2) (3-10). All E2F/DP family proteins contain two highly conserved domains: the sequence-specific DNA-binding domain and the dimerization domain. E2F exhibits strong transcriptional activity when it forms a heterodimer with DP protein. E2F1 was initially identified as a cellular factor required for the transactivation of the adenovirus E2 promoter by the E1A oncoprotein (11). Subsequently, several studies (7,(12)(13)(14) have shown that this transcription factor regulates the timely expression of numerous genes (e.g. cyclin E, CDC2, cyclin A, B-Myb, E2F1, and p107) involved in cell cycle progression as well as several enzymes (e.g. DNA polymerase ␣, thymidine kinase, and dihydrofolate reductase) required for DNA replication.DP-1 was first identified in 1993 as the partner of the E2F family member E2F1 (15, 16). Several studies (17-19) have shown that DP-1 is expressed at high levels in various murine and human tissues. Interestingly, a recent study (20) demonstrated that targeted inactivation of the Dp-1 locus in mice causes severe abnormalities during development of extra-embryonic tissue, which leads to embryonic lethality. This suggests an important role for DP-1 in morphogenesis. Therefore, it is of ...