Pro-inflammatory cytokines induce suppressor of cytokine signaling-3 in human periodontal ligament cells.

INTRODUCTION: Periapical inflammation is initiated by insult to the dental pulp and mediated by inflammatory cytokines in the periodontal tissue. On the other hand, the destruction of tissue can be prevented by the suppression of pro-inflammatory cytokine activity. The balance between these cytokines and their counterregulatory molecules has been suggested to regulate tissue destruction. Suppressors of cytokine signaling (SOCS) proteins are known to suppress inflammatory cytokine signaling via the classic negative feedback loop. However, the mechanism by which they are induced by inflammatory cytokines and regulated during the development of periodontal disease remains to be clarified. We investigated the effects of inflammatory cytokines on SOCS protein expression and their signaling pathways in human periodontal ligament (PDL) cells. METHODS: We examined the effect of inflammatory cytokines on SOCSs expression and its signaling pathway in human PDL cells using reverse transcription- and real-time polymerase chain reaction, Western blot methods. Furthermore, we also examined whether these cytokines-induced SOCS-3 suppress chemokines secretion using ELISA methods. RESULTS: We found that inflammatory cytokines interleukin (IL)-1beta and IL-6 induced expression of SOCS-3 but not that of SOCS-2 in human PDL cells. IL-1beta and IL-6 simultaneously induced IL-8 and monocyte chemoattractant protein-1 secretion in PDL cells, whereas SOCS-3 overexpression suppressed secretion of these chemokines through inhibition of phosphorylation in downstream signaling. CONCLUSION: The results suggest that pro-inflammatory cytokines induced SOCS-3 expression. The SOCS-3 induction suggests playing an important role in negative feedback, suppressing serious destruction of periodontal tissue in apical periodontitis through a chemokine-dependent mechanism.

SOCS-3 inhibits E2F/DP-1 transcriptional activity and cell cycle progression via interaction with DP-1.

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.

Identification and characterization of novel isoforms of human DP-1: DP-1{alpha} regulates the transcriptional activity of E2F1 as well as cell cycle progression in a dominant-negative manner.

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-1alpha and DP-1beta) and characterized their structure and function. DP-1alpha is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1beta is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast two-hybrid and immunoprecipitation assays demonstrated that DP-1alpha binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1beta. 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-1beta. However, such a translocation for DP-1alpha 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-1alpha-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1alpha is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression.