Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

STs were cut into 4 μm sections and fixed in cold acetone for 20 minutes. Endogenous peroxidase was quenched by treatment with 3% H₂O₂ for 5 minutes. STs were next pretreated with 3% goat sera for 1 hour at 37°C before application of primary antibody in 4°C overnight. Indirect immunoperoxidase staining was performed at 37°C for 1 hour. Polyclonal antibody (pAb) rabbit anti-human pFAK, pAb rabbit anti-human pPyk2, pAb rabbit anti-human pSrc, pAb rabbit anti-human pPaxillin and pAb rabbit anti-human pPLCγ were all purchased from Biosource (Camarillo, California, USA) or Cell Signaling Technology (Beverly, Massachusetts, USA), and were used at a concentration of 1 μg/ml. Isotype-specific IgG (rabbit) was used as a negative control. Staining was performed using Vector Elite ABC Kits (Vector, Burlingame, California, USA) and diaminobenzidine (Kirkegaard and Perry, Gaithersburg, Maryland, USA) as a chromogen.

Vascularity was defined as a score as follows: 1, marked decrease in vessels; 2, normal density of vessels; 3, increased density of vessels; 4, marked increase in vessel density, resembling granulation tissue. Inflammation was defined as a score as follows: 1, normal; 2, mildly increased number of inflammatory cells, arrayed as individual cells; 3, moderately increased number of inflammatory cells including distinct clusters (aggregates); 4, marked diffuse infiltrate of inflammatory cells. MΦs were distinguished from fibroblasts based on morphology and CD 11b/c immunoreactivity. Score data were pooled and the mean ± SEM was calculated in each data group [25‐27]. Each of the ST components was graded for immunostaining by a frequency of attaining scale, scored 0–100% where 0% indicates no staining and 100% indicates that all cells were immunoreactive. The number of cells of a given type that reacted with a specific antibody divided by the total number of cells of that given type was defined as the percentage of reactivity. The mean percentage of reactivity was determined for 3 high power fields (HPF) in STs for each cell type and antibody analyzed. Each slide was evaluated by a single blinded pathologist (GKH). Selected sections were analyzed by an additional observer (SS).

RA fibroblasts were isolated from fresh STs by mincing and digesting in a solution of dispase, collagenase and DNase [28]. Cells were used at passage 4 or older, at which time they are a homogeneous population of fibroblasts. Cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) [29]. Mononuclear cells were isolated by Histopaque (Sigma Chemical Co., St. Louis, Missouri, USA) gradient centrifugation. PB monocytes were then isolated from the mononuclear cells by Percoll (Sigma Chemical Co.) gradient centrifugation and countercurrent centrifugal elutriation (Beckman-Coulter, Fullerton, California, USA) [29]. Following adherence, monocytes were differentiated in vitro for 7 days in RPMI containing 20% FBS plus 1 μg/ml polymyxin B sulfate (Sigma Chemical Co). As PB monocytes were isolated from buffy coats by elutriation polymyxin B was added to the media preventatively. The endotoxin levels in RPMI, FBS and PBS as measured by Limulus Amebocyte Lysate (LAL) (QCL-1000; Cambrex Bioscience, Maryland, USA) were below the lowest detectable level of 0.1 endotoxin unit (EU). The endotoxin levels in TNFα and IL1β were lower than 1.0 EU per 1 μg of the cytokine as determined by the LAL method.

RA ST fibroblasts (cultured in DMEM with 10% FBS) and MΦs (cultured in RPMI with 20% FBS) were either untreated or treated with TNFα (10 ng/ml; R&D Systems, Minneapolis, New Mexico, USA) [30] or IL1β (10 ng/ml; R&D Systems) [31] for 0 to 120 min.

Western blot analysis was conducted as previously described [29]. Briefly, 60 μg of each sample was loaded on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes using a semi-dry transblotting apparatus (Bio-Rad, Hercules, California, USA). Nitrocellulose membranes were blocked with 5% nonfat milk in Tris-buffered saline Tween (TBST) buffer-20 mM Tris, 137 mM NaCl, pH 7.6, with 0.1% Tween for 60 min at room temperature. Blots were probed with rabbit anti-pFAK (Tyr 576/577), anti-pPyk2 (Tyr 402), or anti-pSrc (Tyr 527) (Cell Signaling Technology) overnight and after stripping reprobed with rabbit anti-FAK, anti-Pyk2 or anti-Src (Cell Signaling Technology at 1:1000) overnight.

The data was analyzed using Student's t-tests. P values less than 0.05 were considered significant.

As expected, the inflammatory and vascularity scores were higher in RA ST in comparison to OA and NDs. pFAK, was expressed on ST lining cells in RA patients (mean of 15% positive cells) and OA (21%) more than on ND ST lining (1%) (P =< 0.05) (Figure 1). pFAK staining on MΦs was also significantly higher in RA (39%) and OA (25%) compared to ND (4%) (P < 0.05). A few RA patients had positive immunostaining for pFAK on ST endothelial cells and lymphocytes. Unstimulated RA ST fibroblasts expressed pFAK; however, the expression increased with TNFα stimulation at 45 min and stayed upregulated until 120 min (Figure 1e). Similarly, IL1β increased pFAK expression at 30, 45 and 120 min in RA ST fibroblasts (Figure 1f). pFAK was not detected in MΦs with or without TNFα or IL1β stimulation.

pPyk2 is one of the members of the nonreceptor protein tyrosine kinase FAK family and shares approximately 45% sequence homology with FAK. Both proteins are important for integrin-mediated adhesion and osteoclastogenesis [18]. pPyk2 immunostaining on ST lining and MΦs was significantly higher in RA (lining cells = 60% and MΦs = 46%) compared to OA (lining cells = 30% and MΦs = 23%) and ND (lining cells= 17% and MΦs = 10%) (P < 0.05) (Figure 2). However, no difference was detected in pPyk2 lining cells and MΦ immunostaining in OA and ND STs. The relative pattern of pFAK and pPyk2 expression was similar in RA patient MΦs (pFAK = 39%, pPyk2 = 46%); however, pPyk2 was highly expressed on synovial lining (pFAK = 15%, pPyk2 = 60%) compared to pFAK. The relative pattern of pFAK and pPyk2 expression was similar in OA patients' synovial lining (pFAK = 21%, pPyk2 = 30%) and MΦs (pFAK = 25%, pPyk2 = 23%). Interestingly, although pFAK was similarly expressed in RA and OA patients, the percentage of pPyk2 positive cells was significantly higher in RA ST lining and sublining compared to that of OA and ND. Rarely, RA patients showed positive immunostaining for pPyk2 on ST endothelial cells, fibroblasts and lymphocytes. pPyk2 was detected on unstimulated ST fibroblasts, and the expression was further increased by TNFα and IL1β stimulation and stayed upregulated up to 120 min (Figure 2d,e). MΦs did not express pPyk2 at baseline; however, after 30 to 45 min stimulation with TNFα or IL1β a robust level of pPyk2 was detected. In MΦs, pPyk2 remained activated for 90 min subsequent to TNFα or IL1β activation and thereafter markedly decreased (Figure 2f, h).

Integrin αvβ3 activation induces FAK and Pyk2 phosphorylation via Src. Phosphorylation of FAK and Pyk2 results in formation of a signaling complex consisting of signaling molecules including Src and paxillin. Formation of a Pyk2-Src complex and the kinase activity of Src is required for bone resorption by osteoclasts [32]. Src is highly phosphorylated in RA ST lining cells (RA = 68%) and MΦs (RA = 57%) compared to OA (lining cells = 13%, MΦs = 16%) and ND ST (lining cells = 6%, MΦs = 10%) (Figure 3). Although pSrc associated with both FAK and Pyk2 signaling complexes, the expression pattern of pSrc in RA ST lining (pSrc = 68%, pPyk2 = 60%) and sublining (pSrc = 57%, pPyk2 = 46%) was similar to pPyk2, while pSrc immunostaining in OA ST lining (pSrc = 12%, pPyk2 = 30%, pFAK = 21%) and sublining (pSrc = 15%, pPyk2 = 23%, pFAK = 25%) was comparable to both Pyk2 and FAK. RA ST endothelial cells and lymphocytes were occasionally immunopositive for pSrc. Both RA ST fibroblasts and differentiated MΦs expressed pSrc at baseline. pSrc remained activated in RA ST fibroblasts stimulated with TNFα up to 120 min (Figure 3d). In contrast, IL1β induced activation of pSrc no longer than 45 min in RA ST fibroblasts (Figure 3e). In MΦs, both TNFα and IL1β mediated robust activation of pSrc in a time dependent manner up to 120 min (Figure 3f and 3h).

Paxillin is a multidomain adaptor protein that interacts with signaling proteins such as FAK, Pyk2, Src and PLCγ [6, 33]. The phosphorylation of paxillin is modulated by cell adhesion. Paxillin is recruited to the FAK and Pyk2 signaling complex upon integrin αvβ3 ligation in osteoclasts. We found that pPaxillin is expressed on ST lining cells in RA patients (77%) to a significantly higher degree than OA (37%) and ND (12%) (P < 0.05). pPaxillin immunostaining on MΦs was also significantly higher in RA (70%) compared to OA (40%) and ND (14%) (P < 0.05) (Figure 4). Similar to pSrc and pPyk2, paxillin was highly phosphorylated in RA ST lining (pPaxillin = 77%, pSrc = 68%, pPyk2 = 60%) and sublining (pPaxillin = 70%, pSrc = 57%, pPyk2 = 46%). In contrast, pPaxillin immunostaining on OA lining (pPaxillin = 37%, pPyk2 = 30%, pFAK = 21%) and sublining (pPaxillin = 40%, pPyk2 = 23%, pFAK = 25%) was comparable to pFAK and pPyk2. Our findings suggest that the colocalization and activation of FAK, Pyk2, Src and paxillin in RA and OA patient's ST lining and sublining may be important for integrin-mediated signaling.

Upon αvβ3 integrin-mediated adhesion, PLCγ associates with the Pyk2 and FAK signaling complex [6, 33]. M-CSF can also induce association of αvβ3 integrins with PLCγ, PI3K and Pyk2 in a Src-independent manner [33]. The inflammatory and vascularity scores for pPLCγ immunostaining were higher in RA ST in comparison to OA and ND. RA patients most strongly expressed pPLCγ in the lining (67%) and on MΦs (61%) in ST, compared to OA patients (lining = 9%, MΦs = 28%) and ND subjects (lining = 10%, MΦs = 14%)(Figure 5). Interestingly pPLCγ immunostaining was similar on OA and ND MΦs. The positive immunostaining of pPLCγ was comparable to pPyk2 expression on RA ST lining (pPLCγ = 67%, pPyk2 = 60%) and sublining (pPLCγ = 61%, pPyk2 = 46%). Whereas, pPLCγ immunostaining on OA lining (pPLCγ = 9%, pPyk2 = 30%) was lower than that of pPyk2. These results suggest that Pyk2 and its associated signaling protein complex, namely Src, paxillin, and PLCγ are activated on the RA ST lining and MΦs to a greater extent than on OA ST. A few RA patients had positive immunostaining for pPLCγ on fibroblasts and lymphocytes.