Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

Abstract

Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.

Progranulin and Its Receptor Predict Kidney Function Decline in Patients With Type 2 Diabetes

“…Although the exact role of TNFRs is not yet understood, soluble TNFRs may function as decoys for TNFa (36). In the present study, serum PGRN concentrations were correlated mildly with TNFR1 concentrations; however, it should be noted that other research groups have not found any direct physical or functional interaction between PGRN and TNFRs (37,38).…”

Microglial Progranulin: Involvement in Alzheimer’s Disease and Neurodegenerative Diseases

“…Certain studies concluded that the central mechanism of inhibiting inflammation was the binding and antagonism by PGRN of the tumor necrosis factor receptor (TNFR)-1 and TNFR-2 [95,96], thus blocking the action of the proinflammatory cytokine tumor necrosis factor (TNF-α). However, other studies have not replicated these findings [97,98]; this issue is still unresolved [99]. It has been shown that PGRN can block the downstream activity of TNF-α by inhibiting the expression and release of CXCL9, CXCL10 or IL-10 [58,100].…”

Progranulin Oncogenic Network in Solid Tumors

“…Progranulin binds to TNFR1 and TNFR2 on immune cells, mostly macrophages and Tregs, competing with TNF-alpha for receptor binding, thereby inhibiting TNF-alpha pro-inflammatory activity [ 7 ]. It is important to mention to that progranulin interaction with TNFRs remains controversial, since other groups failed to confirm a direct binding of progranulin to TNFRs [ 61 , 62 , 63 ]. These discrepancies might be due to technical differences in the surface plasmon resonance (SPR) experimental approaches used by different groups [ 64 ].…”