Allan S. Pollock

Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney.

The development of progressive glomerulosclerosis in the renal ablation model has been ascribed to a number of humoral and hemodynamic events, including the peptide growth factor, transforming growth factor-beta 1 (TGF-beta 1). An important role has also been attributed to angiotensin II (AII), which, in addition to its hemodynamic effects, can stimulate transcription of TGF-beta 1. We postulated that increased glomerular production of AII, resulting from enhanced intrinsic angiotensinogen expression, stimulates local TGF-beta 1 synthesis, activating glomerular matrix protein synthesis, and leads to sclerosis. Using in situ reverse transcription, the glomerular cell sites of alpha-1 (IV) collagen, fibronectin, laminin B1, angiotensinogen, and TGF-beta 1 mRNA synthesis were determined at sequential periods following renal ablation. The early hypertrophic phase was associated with global, but transient, increases in the mRNA for alpha-1 (IV) collagen. No changes were noted for fibronectin, TGF-beta 1, and angiotensinogen mRNAs. At 24 d after ablation, at which time sclerosis is not evident, endothelial cells, particularly in the dilated capillaries at the vascular pole, expressed angiotensinogen and TGF-beta 1 mRNAs, as well as fibronectin and laminin B1 RNA transcripts. By 74 d after ablation angiotensinogen and TGF-beta 1 mRNAs were widely distributed among endothelial and mesangial cells, and were particularly prominent in regions of evolving sclerosis. These same regions were also notable for enhanced expression of matrix protein mRNAs, particularly fibronectin. All receptor blockade inhibited angiotensinogen, TGF-beta 1, fibronectin, and laminin B1 mRNA expression by the endothelium. We conclude that, as a result of hemodynamic changes, injured or activated endothelium synthesizes angiotensinogen, triggering a cascade of TGF-beta 1 and matrix protein gene expression with resultant development of the segmental glomerular sclerotic lesion.

Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury

Chronic kidney disease (CKD) and failure are problems of increasing importance. Regardless of the primary etiology, CKD is characterized by tubular atrophy, interstitial fibrosis, and glomerulosclerosis. It has been assumed that diminished matrix metalloproteinase (MMP) activity is responsible for the accumulation of the extracellular matrix (ECM) proteins and collagens that typify the fibrotic kidney. Here we demonstrate that transgenic renal proximal tubular epithelial expression of a specific enzyme, MMP‐2, is sufficient to generate the entire spectrum of pathological and functional changes characteristic of human CKD. At the earliest point, MMP‐2 leads to structural alterations in the tubular basement membrane, a process that triggers tubular epithelial‐mesenchymal transition, with resultant tubular atrophy, fibrosis and renal failure. Inhibition of MMP‐2, specifically in the early, prefibrotic stages of disease may offer an additional approach for treatment of these disabling disorders.—Cheng, S., Pollock, A. S., Mahimkar, R., Olson, J. L., Lovett, D. H. Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB J. 20, E1248–E1256 (2006)

Glomerular Mesangial Cell-specific Transactivation of Matrix Metalloproteinase 2 Transcription Is Mediated by YB-1

Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chronic glomerular disease. We have previously demonstrated that MC activation is directly linked to high level expression of the matrix metalloproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Marti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074–15083), the transcription of which is regulated in a tissue-specific fashion. Recent studies (Harendza, S., Pollock, A., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18786–18796) delineated a strong cis-acting enhancer element, designated MMP-2 RE1, within the 5′-flanking region of the rat MMP-2 gene. Gel shift, DNA footprint, and transcriptional analyses mapped the enhancer element to a unique 40-base pair (bp) sequence located at −1322 to −1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-bp enhancer element with MC nuclear extracts yielded a single protein of 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screening of a human MC cDNA expression library with MMP-2 RE1 exclusively yielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the shifted complex. Examination of the MMP-2 RE1 sequence revealed an incomplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted with recombinant YB-1 on DMS protection footprinting analysis. YB-1 protein preferentially bound the single-stranded components of the 40-bp MMP-2 RE1 and, with increasing concentrations, formed multimeric complexes. Co-transfection of YB-1 in MC increased the enhancer activity within the context of the native MMP-2 promoter, while transfection of non-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to transcriptional suppression. This study indicates that YB-1 is a major, cell type-specific transactivator of MMP-2 transcription by glomerular mesangial cells.

Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy

The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc-dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP-9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability.

The prodomain of interleukin 1α interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells

Interleukin 1α (IL‐1α), a 33 kDa precursor, is cleaved releasing the 17 kDa carboxyl‐terminal cytokine IL‐1α to which all of the biological properties of IL‐1α have been attributed. We investigated the potential independent properties of the remaining 16 kDa IL‐1α amino‐terminal propiece by expression in human tumor and primary human cell lines. The IL‐1α propiece produced apoptosis in malignant but not normal cell lines. A minimal fragment comprised of amino acids 55–108 was required for apoptosis. Deletion and mutation studies identified an extended nuclear localization sequence required for nuclear localization, induction of apoptosis and concentration of the IL‐1α propiece in interchromatin granule clusters, concentrations of proteins in the RNA splicing and processing pathways. The IL‐1α propiece interacted with five known components of the RNA splicing/processing pathway, suggesting that the mechanism of action may involve changes in RNA splicing or processing. Expression of the IL‐1α propiece caused a shift in the ratio of Bcl‐Xl/Bcl‐Xs toward the apoptotic direction. Our findings indicate that the IL‐1α propiece induces apoptosis in a range of tumor cells and likely operates through a mechanism involving the RNA processing apparatus and the alternate splicing of apoptosis regulatory proteins.—Pollock, A. S., Turck, J., Lovett, D. H. The prodomain of interleukin‐1 α interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells. FASEB J. 17, 203–213 (2003)

The disintegrin domain of ADAM9: a ligand for multiple β1 renal integrins

Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1 class.

YB-1 ALTERS MT1-MMP TRAFFICKING AND STIMULATES MCF-7 BREAST TUMOR INVASION AND METASTASIS

YB-1 is a member of the cold shock domain family, with complex roles in DNA structure, gene transcription and translation. YB-1 promotes chromosomal instability, and mammary gland transgenic expression induces tumors with 100% penetrance. YB-1 is linked to poor prognosis in breast carcinoma and is a strong predictor of relapse and disease-specific survival. Survival is directly tied to the extent of local invasion and distal metastasis, processes dependent upon the activity of the membrane type I-matrix metalloproteinase, MT1-MMP. Non-invasive MCF-7 breast adenocarcinoma cells were transfected with YB-1/EGFP. YB-1 protein was detected in the invadopodia of cells with a migratory phenotype. There was increased expression of MT1-MMP protein concentrated at the leading edges of motile cells, which were highly invasive in collagen three-dimensional culture. The rates of MT1-MMP protein endocytosis and recycling to the cell surface were elevated in clones expressing higher levels of YB-1 protein. Control MCF-7 cells formed nonfatal, non-invasive, differentiated adenocarcinomas in vivo. MCF-7 cells expressing a twofold increase in YB-1 formed highly anaplastic tumors with local invasion, pulmonary metastases and high lethality. We conclude that YB-1 contributes to the development of an invasive, metastatic breast carcinoma phenotype by enhanced presentation of MT1-MMP at the sites of cellular invasion.

Transfection-mediated Expression of a Dominant cAMP-resistant Phenotype in the Opossum Kidney (OK) Cell Line Prevents Parathyroid Hormone-induced Inhibition of Na-phosphate Cotransport A Protein Kinase-A-mediated Event

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.

The effects of cycloheximide on Na+/H+ antiporter activity in cultured opossum kidney cells

These studies examined the effects of cycloheximide on the Na+/H+ antiporter in cultured opossum kidney cells. The effects of cycloheximide on antiporter activity depended on the basal level of activity. These data suggest that the Na+/H+ antiporter may be regulated by several processes which are sensitive to protein synthesis inhibition.