TNFα and monocyte adhesion differentially induce cathepsins K and V activity
To determine how TNFα and monocyte interactions, individually and cooperatively, regulate cathepsin activity in large artery endothelial cells, we co-cultured human aortic endothelial cells (HAECs) and THP-1 monocytes, as described in the Materials and Methods. TNFα-stimulated mature cathepsin K expression and activity (37 kDa) in HAECs and HAEC/monocyte co-cultures, and also increased cathepsin V expression and activity (35 kDa) by two-fold (; n=3, p<0.05). THP-1 monocytes alone did not stimulate cathepsin K activity, but co-culture with endothelial cells stimulated a 50% increase in cathepsin V activity ( lane 3). TNFα and co-culturing with THP-1 monocytes stimulated a 460% increase in cathepsin V active enzyme compared to HAEC controls ( lane 6; n=3, p<0.05).
Fig 1 TNFα and direct monocyte adhesion induced cathepsin K and V activities in endothelial cell-monocytes co-cultures. Endothelial cells, THP-1 monocytes, and co-cultures were conditioned with 10ng/mL TNFα. Monocytes were allowed to interact (more ...)
In order to ascertain if the increased active cathepsin observed in the co-cultures was mediated by direct monocyte-endothelial cell contacts, paracrine factors, or some combination of both, we implemented a transwell culture system permitting exchange of soluble factors between the cell types, while being physically separated by a 0.22 μm pore size filter. Indirect communication between monocytes and endothelial cells failed to increase cathepsin V activity as high as direct contact cultures; additionally, there was no detectable cathepsin K activity without TNFα stimulation ().
TNFα is sufficient to turn on cathepsin K activity in endothelial cells
To confirm the identity of the apparent TNFα-dependent, 37kDa active band as cathepsin K, HAECs were transfected with CMVSport6 plasmid with cathepsin K gene to drive constitutive overexpression. We achieved 25% transfection efficiency as estimated from parallel transfections with GFP vector with same concentration and protocol (data not shown). Lysates from transfected HAECs were loaded for zymography in the same gel as lysates from HAECs stimulated with TNFα or vehicle, and results are shown in . Transfected HAECs displayed an active band at the same electrophoretic migration distance as that of HAECs stimulated with TNFα, and with greater intensity than control cells confirming the 37kDa band as cathepsin K (). Further confirmation was achieved with an exclusionary cathepsin zymography modification; we previously demonstrated that lowering the pH from 6 to 4 during overnight incubation selects for cathepsin V activity and reduces the cathepsin K signal [17
]. When incubated at pH 4, the upper 37 kDa band intensity diminished in the TNFα-stimulated samples, but cathepsin V (35 kDa) signal remained detectable under both conditions () confirming the upper band as cathepsin K.
Fig 2 TNFα turns on cathepsin K activity in endothelial cells (A) ECs were transfected with cathepsin K gene on pCMVSport6 to drive overexpression. Cell lysates collected from HAECs treated with and without 10ng/mL TNFα, and ECs transfected (more ...)
Next, we sought to confirm that cathepsin K activity in endothelial cells was dependent on TNFα stimulation. HAECs were conditioned for 24 hours in media containing 10 ng/mL TNFα and either 10 μg/mL anti-TNFα antibody or isotype control, and cell lysates were collected. As expected, TNFα stimulated robust cathepsin K activity, while co-incubation with the blocking antibody substantially reduced activity levels (). These data, taken with the observed responses in , indicate that TNFα is sufficient and necessary to stimulate cathepsin K activity in these studies.
Quenched, fluorescent synthetic substrates are commonly used to quantify the activity of cathepsin family members in cells and in vitro
], and we used this method to identify TNFα stimulated cathepsin K activity in situ
as increased fluorescence captured by microscopy. After HAECs were stimulated with TNFα, culture media was replaced with zymography assay buffer containing the cathepsin K cleavable substrate Z-GPR-MβNA (5 μM), and fluorescent images were captured. To select for the cathepsin K activity among other proteases that can cleave this substrate, parallel cultures were inhibited with 5 μM E-64 to block all cathepsin activity or with a protease inhibitor cocktail (10 μM CA-074, 1 mM phenylmethanesulfonylfluoride (PMSF), and 10 mM EDTA to inhibit cathepsin B, serine proteases, and matrix metalloproteinases, respectively) thereby identifying the residual activity as cathepsin K. TNFα stimulation increased total fluorescent intensity, and more importantly, the fluorescence due to cathepsin K seen after incubation with the protease inhibitor cocktail (). E-64 incubation significantly reduced fluorescent intensity as expected, as shown in the picture and indicated by the dashed line on the graph ().
TNFα stimulation and monocyte interactions with endothelial cells increased JNK and Akt phosphorylation
Next, the intracellular signal cascades initiated by TNFα and THP-1 monocyte adhesion, which appeared to have increased cathepsin K and V activities, were investigated at baseline (0 hours), after TNFα stimulation and monocyte binding (4 hours), and six hours of co-culture (10 hours). Co-cultures were maintained for 6 hours instead of 20, to shorten the length of time between stimulation and analysis to quantify the phosphorylated kinase signal before it was quiesced. Cell lysates were analyzed for phosphorylation of Akt, ERK1/2, JNK, and c-Jun using Bioplex technology, and results shown in . JNK and its downstream signaling protein substrate, c-Jun showed the greatest activation in response to TNFα stimulation by 2.8 and 5.3 fold, respectively (, n=3, p<0.01). Akt phosphorylation was significantly increased by TNFα stimulation and monocyte binding (, n=3, p<0.01). There were no changes in ERK 1/2 phosphorylation in any condition for all time points measured ().
Fig 3 TNFα and monocytes interactions increase JNK and Akt phosphorylation. Confluent HAECs and co-cultures were pre-conditioned with 10ng/mL TNFα prior to monocyte adhesion as described earlier. HAEC and co-culture cell lysates were collected (more ...)
JNK inhibition significantly decreased TNFα and THP-1 monocyte induced cathepsin K and V activities
Since TNFα stimulation of HAECs increased cathepsin K and V activities, and JNK and c-Jun were highly activated in response, we next tested the hypothesis that inhibiting JNK pathway would reduce cathepsin K and V activity. Endothelial cells were incubated for 1 hour with the JNK inhibitor SP6000125 (10 μM), followed by stimulation with 10ng/mL TNFα or vehicle for 4 hours, and co-culture with THP-1 monocytes. Inhibition of JNK significantly reduced cathepsin K active enzyme by 49% in HAEC cultures stimulated with TNFα and by 39% in co-cultures stimulated with TNFα (; n=3, p<0.05). In the absence of TNFα stimulation, there was no detectable cathepsin K activity. A similar effect was observed for cathepsin V; JNK inhibition reduced TNFα stimulated active cathepsin V by 60% (n=3, p<.005) in HAECs, by 27% in co-cultures (n=3, p<0.005), and by 81% in TNFα stimulated co-cultures (n=3, p<0.001) ().
Fig 4 Cathepsins K and V activities induced by THP-1 monocytes are significantly reduced by JNK inhibition with SP6000125. (A) HAECs were incubated with or without 10μM of SP6000125 for 1 hour, followed by conditioning with TNFα or vehicle for (more ...)