TNFα, PDGF, and TGFβ synergistically induce synovial lining hyperplasia via inducible PI3Kδ

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enhances the production of inflammatory mediators in monolayer FLSs [12].
Monolayer culture or invasion assays of FLSs are frequently used but have difficulty in the analysis of factors relating to hypertrophic architecture formed by FLSs. Kiener et al. showed that a micromass culture of human FLSs formed lining/sublining architecture resembling synovial tissues, and found that TNF and PDGF contributed to the hypertrophic architecture of intimal lining layer to some extent [13]. However, further mechanisms underlying the hypertrophy of intimal lining driven by FLSs remain to be determined.
In this study, we show that the combination of TNF, PDGF, and TGF synergistically induces obvious hypertrophic architecture in FLS micromass culture, and that inducible PI3K plays a key role in the persistent activation of the PI3K-Akt pathway, and in the formation of hypertrophic FLS architecture.

Isolation of fibroblast-like synoviocytes (FLSs)
Ethical approval for using human samples for this study was granted by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine. Written informed consent was obtained from all study participants. RA was diagnosed in accordance with the criteria of the American College of Rheumatology [14]. FLSs from patients with RA were prepared and cultured in DMEM (Sigma-Aldrich, St. Louis, MO ) supplemented with penicillin (Sigma), streptomycin and 10% FBS (Sigma), as previously described [15].

Micromass culture of FLSs
Micromass cultures of FLSs were prepared, using a process previously reported [13]. Briefly, FLSs were suspended in ice-cold Matrigel Matrix (BD Biosciences, Bedford, MA) at a density of 5  10 6 cells/ml. Droplets (40 l) of the suspension were placed onto poly-2-hydroxyethylmethacrylate (poly-HEMA; Sigma)-coated culture dishes. Gelation was allowed to occur for 45 min at 37C. The gel was then overlaid with culture medium. The floating 3-D culture was maintained for 2-3 weeks with a medium change twice a week.
For stimulation experiments, FLS micromasses were cultured in basal medium containing 10 ng/ml of tumor necrosis factor (TNF), 10 ng/ml of platelet-derived growth factor (PDGF), and/or 10 ng/ml of transforming growth factor (TGF) (each from Pepro Tech; Rocky Hill, NJ).

Histological analysis
Micromasses were processed as 20-m-thick frozen sections and stained with hematoxylin and eosin. Light microscopic images were taken using ECLIPSE 80i (Nikon, Japan) with a 10 dry lens. The area of the intimal lining layer, including villous structures intruding toward the center of the micromass, was measured using ImageJ (NIH).

Cell proliferation assay
FLSs at 50% confluent were cultured for 5 days with or without indicated cytokines. Cell proliferation was measured with CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) according to manufacturer's protocol. Western blot analysis  PI3K was upregulated in FLS micromasses cultured in the TPT condition.
In the hyperplastic synovium of RA, various molecules are elevated and contribute to the pathogenesis of RA. Metalloproteinases, including MMP3, contribute to cartilage erosion and tendon rupture by degenerating the collagen matrix [17][18][19].
Cadherin-11 plays a crucial role in synovial structure by supporting cell-to-cell junctions. In the micromass organ culture, the expression of MMP3 was apparently correlated with the addition of TNF ( Fig. 2A), whereas Cadherine-11 was expressed most in the TPT condition (Fig. 2B). The elevation of PI3K is also reported in RA synovium [19]. Among the isoforms of PI3K, the expression of PI3K significantly We stimulated monolayer FLSs with the TPT condition and investigated the activation of the PI3K-Akt pathway. As previously reported, TNF did not enhance the phosphorylation of Akt, whereas the other conditions, including PDGF alone, the combination of TNFα and PDGF, and the TPT condition, each similarly increased phosphorylated Akt (P-Akt) 6 h after the stimulation (Fig. 3B). The TPT condition also increased the amount of P-Akt 24 h after the stimulation compared with other conditions (Fig. 3B). The time course of P-Akt, measured by cell-ELISA, showed that its increase under stimulation with PDGF was similar to that under stimulation with TNF and PDGF together, and with the TPT condition for the first 6 h. In the subsequent 18 h, the amount of P-Akt under the condition of PDGF, and with the combination of PDGF and TNF, gradually decreased, but continued to increase in the TPT condition (Fig. 3C). The amount of P-Akt in the TPT condition was significantly higher than that of other conditions at both 24 h and 48 h after stimulation ( Fig. 3D and E). These data indicate that stimulation of monolayer FLSs with the TPT condition enhances PI3K and persistently activates the PI3K-Akt pathway.
Knockdown of PI3K in monolayer FLSs attenuated the activation of the PI3K-Akt pathway induced by the TPT condition.
To determine the involvement of PI3K in the activation of the PI3K-Akt pathway, we knocked down the expression of PI3K. First, we confirmed that transfection of siPI3K to monolayer FLSs specifically downregulated the mRNA of PI3K but not that of PI3K, nor PI3K (Fig. 4A). PI3K siRNA significantly attenuated the proliferation of monolayer FLSs (Fig. 4B) and the phosphorylation of Akt induced by the TPT condition at each time point during 48 h (Fig. 4C). These results indicate that inducible PI3K of FLSs plays a crucial role in FLS proliferation and in the persistent activation of the PI3K-Akt pathway induced by the TPT condition.
PI3K is involved in hyperplastic synovial lining.
We have shown that the TPT condition induces hypertrophy of the intimal lining layer and also enhances the expression of PI3K in Figures 1 and 2. To determine whether PI3K is functionally involved in the formation of hypertrophic synovial lining, PI3K-knocked down FLSs were conducted 3-D culture under the TPT condition. We confirmed that the knockdown of PI3K persisted for at least 10 days (data not shown).
Knockdown of PI3K significantly inhibited the thickening of the intimal lining layer induced by the TPT condition ( Fig. 4D and E). Thus, these results collectively suggest that the TPT condition induces hypertrophic architecture in synovium by elevating the expression of PI3K, and then activating the PI3K-Akt pathways.

Discussion
Clinical reports showed that synovial hyperplasia of RA contributed to the deformation of joints [2].  [20][21][22]. TGF is a kind of growth factor, but unlike PDGF, it has inhibitory effects and proliferative effects depending on the target cells [9][10][11]23]. In monolayer FLSs, the combination of PDGF and TGF synergistically enhances the production of MMP3, IL-8, and MIP1a induced by TNF [12], and activates the PI3K-Akt pathway [24]. In addition, we have shown that this combination also has a synergistic effect in the thickening of the synovial lining. Long-lasting activation of the PI3K-Akt pathway (Fig. 3C) might contribute to the architectural change in micromasses of FLSs.
PI3K has constitutive isoforms of PI3K and PI3K, and inducible isoforms of PI3K and PI3K. A deficiency of the constitutive isoforms, PI3K or PI3K, is embryonic lethal [25,26], implying the difficulty of treating RA by targeting these molecules. On the other hand, PI3K is preferentially expressed by lymphoid cells [27] and RA hypertrophic synovium [19], and a deficiency of PI3K does not affect the lifespan, and ameliorates arthritis in mice [28,29]. Although B cells were considered to be the most responsible cells for this amelioration, our study showed that inducible PI3K of FLSs is also involved in the activation of the PI3K-Akt pathway and synovial lining hyperplasia (Fig. 4B and 4C).
In this study, we addressed the signaling pathway of TNFα, PDGF and TFGβ using monolayer FLSs. MMP3 and PI3Kδ showed similar expression in both monolayer and micormass cultures, suggesting that the analysis of the PI3K-Akt pathway using monolayer FLSs is reasonable. On the other hand, Cadherin-11 failed to increase in monolayer FLSs under TPT condition. The discrepancy in Cadherin-11 might be partly attributed to a correlation between Cadherin-11 and adherens junction at the site of cell-cell contact [30]. The TPT condition enhanced the proliferation of monolayer FLSs most (Fig. 1J), whereas the difference of proliferation between cytokine conditions was not so obvious as expected from the results of micromass cultures (Fig. 1I). Furthermore, TNFα apparently contributes most to the proliferation of FLS compared to PDGF and TGFβ (Fig. 1J), whereas least to synovial hyperplasia (Fig. 1I). This indicates that Conflict of interest None.   Data are shown as mean ± SEM of triplicate. * = P < 0.05 in two-way ANOVA.