Barbara J. Ballermann

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloprotein‐ases (MMP) has been studied extensively due to the fundamental roles these zinc‐endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP‐2 contains 29 potential phosphorylation sites. Mass spectrometryreveals that at least five of these sites are phosphorylated in hrMMP‐2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP‐2 immunoreactivity on 2D immuno‐blots consistent with phosphorylation, and purified MMP‐2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP‐2 from HT1080 cell‐conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP‐2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP‐2 significantly affects its enzymatic properties. Consistent with this, dephos‐phorylation of MMP‐2 immunoprecipitated from HT1080 conditioned medium with alkaline phospha‐tase significantly increases its activity. We conclude that MMP‐2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.—Sariahmetoglu, M., Crawford, B. D., Leon, H., Sawicka, J., Li, L., Ballermann, B. J., Holmes, C., Berthiaume, L. G., Holt, A., Sawicki, G., Schulz, R. Regulation of matrix metalloproteinase‐2 activity by phosphorylation. FASEB J. 21, 2486–2495 (2007)

Glomerular endothelium: a porous sieve and formidable barrier.

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.

Contribution of the Endothelium to the Glomerular Permselectivity Barrier in Health and Disease

Background: The endothelium that lines glomerular capillaries shares many properties with endothelial cells in general, but unlike most endothelial cells, it is extremely flat and densely perforated by transendothelial cell pores, the fenestrae. Until recently, it was believed that the fenestrae allow free passage of large proteins, and that the glomerular endothelium contributes little to the permselectivity of the glomerular capillary wall. Methods: Key studies addressing the nature of the glomerular capillary endothelium and its contribution to glomerular permselectivity were reviewed. Results: Glomerular endothelial cell flattening and fenestrae formation requires signals from differentiated podocytes, and from the glomerular basement membrane. Deletion of VEGF-A from podocytes prevents flattening and fenestration of glomerular endothelium. Application of VEGF-A to endothelial cells in vivo stimulates fenestrae formation, and neutralization of VEGF-A by soluble VEGF receptor 1 (sFlt-1) or anti-VEGF antibodies results in loss of glomerular fenestrae, and proteinuria. Neutralizing TGF-β1 antibodies, deletion of laminin α3 in mice or laminin β3 in humans cause similar defects. The glomerular endotheliosis lesion of pre-eclampsia is due to the placenta-derived inhibitors sFlt-1 and sEndoglin, which block the VEGF-A/VEGF receptor and TGF-β/endoglin signaling, respectively, causing the loss of glomerular endothelial cell fenestrae, cell swelling and proteinuria. The glomerular endothelium is covered by a glycocalyx that extends into the fenestrae and by a more loosely associated endothelial cell surface layer of glycoproteins. Mathematical analyses of functional permselectivity studies have concluded that the glomerular endothelial cell glycocalyx and its associated surface layer account for the retention of up to 95% of proteins within the circulation. Furthermore, the fenestrae are critical for the maintenance of the high hydraulic conductivity of the glomerular capillary wall, and their loss results in a reduction in the glomerular filtration rate. Conclusions: Loss of GFR and proteinuria can result from glomerular endothelial cell injury.

A human glomerular SAGE transcriptome database

BackgroundTo facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach.DescriptionThe database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen.ConclusionThe database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.

Phosphorylation of Rac1 T108 by extracellular signal-regulated kinase in response to epidermal growth factor: a novel mechanism to regulate Rac1 function.

ABSTRACT Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, 183KKRKRKCLLL192 (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1s role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.

Tipping the balance from angiogenesis to fibrosis in CKD

Chronic progressive renal fibrosis leads to end-stage renal failure many patients with chronic kidney disease (CKD). Loss of the rich peritubular capillary network is a prominent feature, and seems independent of the specific underlying disease. The mechanisms that contribute to peritubular capillary regression include the loss of glomerular perfusion, as flow-dependent shear forces are required to provide the survival signal for endothelial cells. Also, reduced endothelial cell survival signals from sclerotic glomeruli and atrophic or injured tubule epithelial cells contribute to peritubular capillary regression. In response to direct tubular epithelial cell injury, and the inflammatory reaction that ensues, capillary pericytes dissociate from their blood vessels, also reducing endothelial cell survival. In addition, direct inflammatory injury of capillary endothelial cells, for instance in chronic allograft nephropathy, also contributes to capillary dropout. Chronic tissue hypoxia, which ensues from the rarefaction of the peritubular capillary network, can generate both an angiogenic and a fibrogenic response. However, in CKD, the balance is strongly tipped toward fibrogenesis. Understanding the underlying mechanisms for failed angiogenesis in CKD and harnessing endothelial-specific survival and pro-angiogenic mechanisms for therapy should be our goal if we are to reduce the disease burden from CKD.

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity.

TIMAP promotes Angiogenesis by suppressing PTEN-mediated Akt inhibition in Human Glomerular Endothelial Cells

The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 ± 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2-deoxyuridine incorporation by 38 ± 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAP-depleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN.

Clustered PI(4,5)P2 accumulation and ezrin phosphorylation in response to CLIC5A

ABSTRACT CLIC5A (encoded by CLIC5) is a component of the ezrin–NHERF2–podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] reporter RFP–PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA–PIP5K&agr; colocalized with GFP–CLIC5A and was pulled from cell lysates by GST–CLIC5A, and silencing of endogenous PIP5K&agr; abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5K&agr;, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2-generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.

Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP

TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP-PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cβ. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4-1.2 nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cβ effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cβ activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.