TonEBP levels were increased in OA cartilage and 29-kDa FN-f-treated chondrocytes
To investigate whether the level of TonEBP is related to the pathogenesis of OA, TonEBP expression was compared between normal and OA cartilage. The mRNA levels of TonEBP in OA cartilage were significantly increased compared with normal cartilage (Fig. 1A).
Next, we examined whether 29-kDa FN-f influences TonEBP expression at the mRNA and protein levels. TonEBP mRNA was significantly upregulated from 6 h after treatment with 29-kDa FN-f in a time-dependent manner (Fig. 1B). Western blot analysis showed that the TonEBP protein significantly increased after 6 h of treatment with 29-kDa FN-f (Fig. 1C). Notably, the TonEBP mRNA level also increased in control chondrocytes in a time-dependent manner; however, this increase was not accompanied by an increase in protein expression. Immunofluorescence showed that TonEBP was predominantly localized in the cytoplasm in untreated chondrocytes (Fig. 1D). Treatment with 29-kDa FN-f increased the nuclear localization of TonEBP to a degree comparable with that observed in a positive control induced by hyperosmolarity (NaCl, 110 mM) (Fig. 1D). These results demonstrate that expression of TonEBP increased in OA and 29-kDa FN-f-treated chondrocytes.
TonEBP influences 29-kDa FN-f-dependent expression of MMPs
29-kDa FN-f induces MMP expression via the TLR-2/myeloid differentiation primary response 88 signaling pathway 13. To investigate whether TonEBP is involved in 29-kDa FN-f-induced MMP expression, knockdown of TonEBP was performed by transfection with an siRNA targeting TonEBP (si-TonEBP). 29-kDa FN-f highly increased expression of MMPs 1, 3, and 13 at both the mRNA and protein levels, whereas TonEBP silencing significantly reduced 29-kDa FN-f-stimulated MMP expression (Fig. 2A–C). These results demonstrate that TonEBP is a regulator of 29-kDa FN-f-induced MMP expression.
Activation of mitogen-activated protein kinase (MAPK)/NF-κB signaling via PLC-γ contributes to 29-kDa FN-f-dependent nuclear translocation of TonEBP
The PLC-γ/PKC axis activates several signaling pathways to regulate the expression of ECM-related genes, including MMP-13 and type II collagen (Col II), in human OA chondrocytes 16. PLC-γ hydrolyzes phosphatidylinositol biphosphate to diacylglycerol and inositol 1,4,5-trisphosphate, which in turn allow activation of PKC and intracellular mobilization of Ca2+, respectively 17. To elucidate whether PLC-γ activation is involved in 29-kDa FN-f-dependent expression of TonEBP, chondrocytes were treated with 29-kDa FN-f in combination with U73122 (1 µM). First, inhibition of PLC-γ significantly suppressed 29-kDa FN-f-induced expression of MMPs 1, 3, and 13 (Fig. 3A). In addition, activation of downstream signaling molecules of PLC-γ, including PKC, p38, ERK, JNK, and Iκ-Bα, was rapidly increased at 15 min and highly maintained until 60 min after treatment with 29-kDa FN-f (Fig. 3B). Our data showed that the PLC-γ signaling axis was related to expression of MMPs and accompanied by activation of MAPK/NF-κB signaling induced by 29-kDa FN-f.
Next, activation (nuclear translocation) of TonEBP protein was examined 30 min after treatment with 29-kDa FN-f in the presence of U73122 (1 µM) for 2 h. Both 29-kDa FN-f-dependent nuclear translocation and cytoplasmic expression of TonEBP were reduced by addition of U73122 (Fig. 3C). In addition, JNK/ERK/p38 MAPK and Iκ-Bα inhibitors significantly suppressed TonEBP nuclear translocation as well as cytoplasmic expression (Fig. 3D). Therefore, these results demonstrate that 29-kDa FN-f promotes nuclear localization of TonEBP via the PLC-γ/PKC/MAPK and NF-κB signaling pathways.
TLR-2 is responsible for 29-kDa FN-f-dependent activation of TonEBP
We previously demonstrated that 29-kDa FN-f signals via TLR-2 to mediate catabolic and anabolic responses 13,18. To examine the role of TLR-2 in 29-kDa FN-f-mediated TonEBP expression, chondrocytes were transfected with an siRNA targeting TLR-2 (si-TLR-2), followed by treatment with 29-kDa FN-f. TLR-2 knockdown significantly decreased 29-kDa FN-f-induced TonEBP expression at both the mRNA and protein levels (Fig. 4A, B). Furthermore, silencing of TLR-2 significantly decreased nuclear accumulation of TonEBP as well as its cytoplasmic expression (Fig. 4C). In turn, knockdown of TLR-2 apparently abolished 29-kDa FN-f-stimulated activation of PKC, p38 MAPK, and Ik-Bα (Fig. 4D). These results demonstrate that 29-kDa FN-f signals via TLR-2 to induce TonEBP expression and its nuclear translocation.
29-kDa FN-f modulated expression of osmoregulatory genes and voltage-dependent calcium channels (VDCCs) via TLR-2/TonEBP signaling
TonEBP regulation and downstream signaling differ according to the stimulus. Hyperosmolarity (NaCl) increases cellular expression and nuclear translocation of TonEBP, which induces the expression of osmoregulatory genes and heat-shock proteins, leading to prevention of cellular damage, whereas TNF-α induces nuclear accumulation of TonEBP but not expression of osmoregulatory genes 12. We investigated whether 29-kDa FN-f induces expression of osmoregulatory genes, including taurine transporter (TauT/SLC6A6), sodium/myo-inositol cotransporter (SMIT/SLC5A3), and aldose reductase (AR/AKR1B1). As expected, osmoregulatory genes were highly upregulated from 6 h after stimulation with NaCl, persisting for 72 h (Fig. 5A). Interestingly, osmoregulatory genes were slightly but significantly induced by treatment with 29-kDa FN-f for 24 and 48 h, although at a much lower level than that induced by NaCl treatment (Fig. 5A). These data show that 29-kDa FN-f affected the expression of osmoregulatory genes, although its induction was weak and delayed. We examined whether 29-kDa FN-f induces expression of TauT, SMIT, and AR via TLR-2/TonEBP. TLR-2- or TonEBP-silenced chondrocytes were exposed to 29-kDa FN-f for 24 h. PCR analysis showed that increased expression of TauT, SMIT, and AR by 29-kDa FN-f were significantly suppressed in TLR-2- and TonEBP-knockdown cells (Fig. 5B). TLR-2- and TonEBP knockdown also decreased TauT, SMIT, and AR in untreated chondrocytes. These results demonstrate that 29-kDa FN-f can induce osmoregulatory gene expression via the TLR-2/TonEBP signaling pathway.
In response to osmolarity, expression of aquaporin 2, a tonicity-sensitive water channel, is regulated in a TonEBP-dependent, but CaN pathway-independent manner, in NP chondrocytes, indicating that they can readily respond to an applied stimulus such as hyperosmolarity via regulation of channel expression 19. We examined whether 29-kDa FN-f could alter the expression of ion channels, including VDCCs (Cav1.2 and Cav1.3), aquaporin-1 (AQP-1), acid-sensing ion channel (ASIC), transient receptor potential cation channel subfamily V 4 (TRPV4), and purinergic receptor P2X 7 (P2RX7). The expression of VDCCs and AQP-1 was significantly increased by treatment with 29-kDa FN-f at 24 h and remained high until 48 h. Their expression levels were also increased in untreated chondrocytes after 72 h of culture (Fig. 6A). By contrast, the expression levels of ASIC, TRPV4, and P2RX7 were not affected by 29-kDa FN-f (Fig. 6A). Thus, these data demonstrate that prolonged treatment with 29-kDa FN-f modulates expression of VDCCs and AQP-1. To examine whether TLR-2/TonEBP signaling influences the expression of VDCCs and AQP-1, TLR-2 in chondrocytes was knocked down by transfection with si-TLR-2 and si-TonEBP. 29-kDa FN-f-induced expression of VDCCs, and AQP-1 was significantly suppressed by silencing of TLR-2 and TonEBP (Fig. 6B), indicating that expression of VDCCs and AQP-1 was significantly increased by 29-kDa FN-f via the TLR-2/TonEBP signaling pathway. These results demonstrate that 29-kDa FN-f regulates expression of osmoregulatory genes and channels related to osmolarity via TLR-2/TonEBP.
29-kDa FN-f-induced MMP expression is dependent on Ca2+/calmodulin (CaM)/calcineurin (CaN) signaling.
In response to stimuli, activation of PLC and generation of inositol 1,4,5-trisphosphate allow rapid transient release of Ca2+ from endoplasmic reticulum (ER) intracellular stores, which activates CaN, a Ca2+/CaM-activated phosphatase, subsequently leading to dephosphorylation and nuclear localization of NFAT proteins. In addition, diacylglycerol, another second messenger produced by PLC, activates PKC in several signaling pathways 20,21. To confirm whether 29-kDa FN-f-induced MMP expression was altered following intracellular calcium perturbation, 29-kDa FN-f-induced MMP expression was measured in the presence of BAPTA or TG. BAPTA-induced depletion of intracellular Ca2+, and TG-induced depletion of ER Ca2+ storage significantly suppressed 29-kDa FN-f-induced expression of MMPs 1, 3, and 13 (Fig. 7A and B), showing that persistent perturbation of Ca2+ inhibits 29-kDa FN-f-induced MMP expression. These results suggest that 29-kDa FN-f can regulate MMP expression via a Ca2+-dependent mechanism. To investigate whether 29-kDa FN-f-induced MMP expression is dependent of CaM/CaN signaling, we first measured the expression of CaM and CaN following exposure to 29-kDa FN-f. Expression of both CaM and CaN was precipitously increased from 48 h after treatment with 29-kDa FN-f (Fig. 7C). CaM and CaN were also increased in the control culture after 72 h. 29-kDa FN-f significantly increased expression of MMPs 1, 3, and 13 at both the mRNA and protein levels (Fig. 7D–F). Silencing of CaM and CaN significantly suppressed 29-kDa FN-f-stimulated expression of MMPs 1, 3, and 13 (Fig. 7D). These results suggest that in addition to the TonEBP signaling pathway, the CaM/CaN signaling pathway can at least partially affect 29-kDa FN-f-induced MMP expression as well.
We next measured the change in the intracellular Ca2+ concentration induced by 29-kDa FN-f using the Fluo-8 Calcium Assay kit. Intracellular influx of Ca2+ was similar between chondrocytes treated with PBS and those treated with 29-kDa FN-f (Fig. 7G). The Ca2+ ionophore A23187, which was used as a positive control, significantly increased intracellular Ca2+ concentrations in both 29-kDa FN-f- and PBS-treated cells (Fig. 7G), indicating that 29-kDa FN-f did not directly induce Ca2+ influx.