IHG-2, a mesangial cell gene induced by high glucose, is human gremlin. Regulation by extracellular glucose concentration, cyclic mechanical strain, and transforming growth factor-beta1

We used cloning in silico coupled with polymerase chain reaction to demonstrate that IHG-2 is part of the 3'-untranslated region of gremlin, a member of the DAN family of secreted proteins that antagonize the bioactivities of members of the transforming growth factor (TGF)-beta superfamily. Mesangial cell gremlin mRNA levels were induced by high glucose, cyclic mechanical strain, and TGF-beta1 in vitro, and gremlin mRNA levels were elevated in the renal cortex of rats with streptozotocin-induced diabetic nephropathy in vivo. gremlin expression was observed in parallel with induction of bone morphogenetic protein-2 (BMP-2), a target for gremlin in models of cell differentiation. Together these data indicate that (a) IHG-2 is gremlin, (b) gremlin is expressed in diabetic nephropathy in vivo, (c) both glycemic and mechanical strain stimulate mesangial cell gremlin expression in vitro, (d) high glucose induces gremlin, in part, through TGFbeta-mediated pathways, and (e) Gremlin is a potential endogenous antagonist of BMPs within a diabetic glomerular milieu.     

Lipoxin A4 antagonizes the mitogenic effects of leukotriene D4 in human renal mesangial cells. Differential activation of MAP kinases through distinct receptors

The lipoxygenase-derived eicosanoids leukotrienes and lipoxins are well defined regulators of hemeodynamics and leukocyte recruitment in inflammatory conditions. Here, we describe a novel bioaction of lipoxin A(4) (LXA(4)), namely inhibition of leukotriene D(4) (LTD(4))-induced human renal mesangial cell proliferation, and investigate the signal transduction mechanisms involved. LXA(4) blocked LTD(4)-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity in parallel to inhibition of LTD(4)-induced mesangial cell proliferation. Screening of a human mesangial cell cDNA library revealed expression of the recently described cys-leukotriene(1)/LTD(4) receptor. LTD(4)-induced mesangial cell proliferation required both extracellular-related signal regulated kinase (erk) and PI 3-kinase activation and may involve platelet-derived growth factor receptor transactivation. LTD(4)-stimulated the MAP kinases erk and p38 via a pertussis toxin (PTX)-sensitive pathway dependent on PI 3-kinase and protein kinase C activation. On screening a cDNA library, mesangial cells were found to express the previously described LXA(4) receptor. In contrast to LTD(4), LXA(4) showed differential activation of erk and p38. LXA(4) activation of erk was insensitive to PTX and PI 3-kinase inhibition, whereas LXA(4) activation of p38 was sensitive to PTX and could be blocked by the LTD(4) receptor antagonist SKF 104353. These data suggest that LXA(4) stimulation of the MAP kinase superfamily involves two distinct receptors: one shared with LTD(4) and coupled to a PTX-sensitive G protein (G(i)) and a second coupled via an alternative G protein, such as G(q) or G(12), to erk activation. These data expand on the spectrum of LXA(4) bioactions within an inflammatory milieu.     

Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages

The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (Mphi) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators, such as lipoxins, act as potent regulators (nanomolar range) of the phagocytic clearance of apoptotic cells. In this study, we have investigated the intriguing possibility that apoptotic cells release signals that promote their clearance by phagocytes. We report that conditioned medium from apoptotic human polymorphonuclear neutrophils (PMN), Jurkat T lymphocytes, and human mesangial cells promote phagocytosis of apoptotic PMN by Mphi and THP-1 cells differentiated to a Mphi-like phenotype. This prophagocytic activity appears to be dose dependent, sensitive to the caspase inhibitor zVAD-fmk, and is associated with actin rearrangement and release of TGF-beta1, but not IL-8. The prophagocytic effect can be blocked by the formyl peptide receptor antagonist Boc2, suggesting that the prophagocytic factor(s) may interact with the lipoxin A(4) receptor, FPRL-1. Using nanoelectrospray liquid chromatography mass spectrometry and immunodepletion and immunoneutralization studies, we have ascertained that annexin-1 and peptide derivatives are putative prophagocytic factors released by apoptotic cells that promote phagocytosis of apoptotic PMN by M[phi] and differentiated THP-1 cells. These data highlight the role of annexin-1 and peptide derivatives in promoting the resolution of inflammation and expand on the therapeutic anti-inflammatory potential of annexin-1.     

The Lipoxin A4 receptor is coupled to SHP-2 activation: implications for regulation of receptor tyrosine kinases

Mesangial cell proliferation is pivotal to the pathology of glomerular injury in inflammation. We have previously reported that lipoxins, endogenously produced eicosanoids with anti-inflammatory and pro-resolution bioactions, can inhibit mesangial cell proliferation in response to several agents. This process is associated with elaborate receptor cross-talk involving modification receptor tyrosine kinase phosphorylation (McMahon, B., Mitchell, D., Shattock, R., Martin, F., Brady, H. R., and Godson, C. (2002) FASEB J. 16, 1817-1819). Here we demonstrate that the lipoxin A(4) (LXA(4)) receptor is coupled to activation and recruitment of the SHP-2 (SH2 domain-containing tyrosine phosphatase-2) within a lipid raft microdomain. Using site-directed mutagenesis of the cytosolic domain of the platelet-derived growth factor receptor beta (PDGFRbeta), we report that mutation of the sites for phosphatidylinositol 3-kinase (Tyr(740) and Tyr(751)) and SHP-2 (Tyr(763) and Tyr(1009)) recruitment specifically inhibit the effect of LXA(4) on the PDGFRbeta signaling; furthermore inhibition of SHP-2 expression with short interfering RNA constructs blocked the effect of LXA(4) on PDGFRbeta phosphorylation. We demonstrate that association of the PDGFRbeta with lipid raft microdomains renders it susceptible to LXA(4)-mediated dephosphorylation by possible reactivation of oxidatively inactivated SHP-2. These data further elaborate on the potential mechanisms underlying the anti-inflammatory, proresolution, and anti-fibrotic bioactions of lipoxins.     

DNA heteroduplex analysis of the relation between bacteriophages phiX174 and S.13

When observed in the electron microscope using the isodenaturing methods of Davis &; Hyman (1971), only one small segment (4.7 ± 1.9%) of the DNA of phage φX174 is highly homologous with phage S.13 DNA; the rest is partially homologous with an over-all average 36% base mismatch. The two phage DNA molecules appear to be identical in length and have no regions of complete base non-homology. The phage-coded proteins were compared by electrophoresis on slab polyacrylamide gels and only one of the S.13 coded proteins migrated identically with its φX174 counterpart. The other eight S.13 coded proteins varied in size from their φX174 counterparts by +4.6% to ?6.0% (± ten amino acid residues). The relevance of these data to the complementation and recombination between these two phages is discussed.