Abstract
Our previous studies showed that urinary Tamm–Horsfall glycoprotein (THP) potently enhanced polymorphonuclear neutrophil (PMN) phagocytosis. However, the domain structure(s), signaling pathway and the intracellular events responsible for THP-enhanced PMN phagocytosis remain to be elucidated. THP was purified from normal human urine. The human promyelocytic leukemia cell line HL-60 was induced to differentiate into PMNs by all-trans retinoid acid. Pretreatment with different MAPK and PI3K inhibitors was used to delineate signaling pathways in THP-enhanced PMN phagocytosis. Phosphorylation of molecules responsible for PMN phagocytosis induced by bacterial lipopolysaccharide (LPS), THP, or human recombinant epidermal growth factor (EGF) was evaluated by western blot. A p38 MAPK inhibitor, SB203580, effectively inhibited both spontaneous and LPS- and THP-induced PMN phagocytosis. Both THP and LPS enhanced the expression of the Rho family proteins Cdc42 and Rac that may lead to F-actin re-arrangement. Further studies suggested that THP and EGF enhance PMN and differentiated HL-60 cell phagocytosis in a similar pattern. Furthermore, the EGF receptor inhibitor GW2974 significantly suppressed THP- and EGF-enhanced PMN phagocytosis and p38 and ERK1/2 phosphorylation in differentiated HL-60 cells. We conclude that EGF receptor-dependent signaling may be involved in THP-enhanced PMN phagocytosis by activating Rho family and MAP kinase.
Highlights
Urinary Tamm–Horsfall glycoprotein (THP) or uromodulin is an 80–90 kDa GPI-anchored protein synthesized by renal tubular cells in the thick ascending limb of Henle’s loop [1]
Our previous reports demonstrated that THP potently enhanced polymorphonuclear neutrophil (PMN) phagocytosis [22,24,25]
RhoA expression decreased on LPS stimulation and showed no change upon THP stimulation (Figure 5C). These results suggest that THP-enhance PMN phagocytosis depends on Cdc42 and Rac, which is somewhat different from LPS
Summary
Urinary Tamm–Horsfall glycoprotein (THP) or uromodulin is an 80–90 kDa GPI-anchored protein synthesized by renal tubular cells in the thick ascending limb of Henle’s loop [1]. The minor O-linked carbohydrate side chains in THP exhibit binding affinities to different protein molecules [15]. These THP-binding proteins include IL-1 [16], TNF-α [17], immunoglobulin light chains [18], and the complement components 1 and 1q [19]. Contradictory to the results of Easton et al [15], we noted that the intact protein-core structure, rather than the carbohydrate side chains, was responsible for THP-enhanced PMN phagocytosis [25]. Intracellular events responsible for THP-enhanced PMN phagocytosis remain unclear. Functional assessments and THP-transduced signaling pathways were investigated to explore the molecular basis of THP-enhanced PMN phagocytosis
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