Grande, Joseph P.

Abstract The homologous proteins p68 and p72 are members of the DEAD box family of RNA helicases. Here, we show that expression of both of these helicases strongly increases during the polyp→adenoma→adenocarcinoma transition in the colon. Furthermore, p68 and p72 form complexes with β-catenin and promote the ability of β-catenin to activate gene transcription. Conversely, simultaneous knockdown of p68 and p72 leads to reduced expression of the β-catenin–regulated genes, c-Myc, cyclin D1, c-jun, and fra-1, all of which are proto-oncogenes. Moreover, transcription of the cell cycle inhibitor p21WAF1/CIP1, whose expression is suppressed by c-Myc, is enhanced on p68/p72 knockdown. Thus, p68/p72 may contribute to colon cancer formation by directly up-regulating proto-oncogenes and indirectly by down-regulating the growth suppressor p21WAF1/CIP1. Accordingly, knockdown of p68 and p72 in colon cancer cells inhibits their proliferation and diminishes their ability to form tumors in vivo. Altogether, these results suggest that p68/p72 overexpression is not only a potential marker of colon cancer but is also causally linked to this disease. Therefore, p68 and p72 may be novel targets in the combat against colon cancer. [Cancer Res 2007;67(16):7572–8]

Abstract PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.

Dyslipidemia often accompanies and accelerates renal disease, partly by promoting fibrosis. However, the mechanisms mediating this effect are unclear. We hypothesized that hypercholesterolemia modulates several interlinked pathways that promote deposition and blunt degradation of extracellular matrix, and that these could be manipulated by reversal of hypercholesterolemia. Fourteen pigs were fed a 16-week 2% high-cholesterol diet (HC-HC; n=7) or normal diet (n=7), whereas in 7 others, a 10-week HC was followed by a 6-week normal diet (HC-N). Renal endothelial function was assessed in vivo with electron-beam computed tomography, and renal tissue was then studied ex vivo using Western blot, real-time quantitative polymerase chain reaction, gelatin zymography, and immunostaining. HC-HC kidneys showed endothelial dysfunction, accompanied by increased intrarenal oxidative stress, inflammation, activation of the endothelin and transforming-growth factor-β systems, and decreased matrix metalloproteinase expression and activity. Accordingly, HC-HC kidneys showed increased collagen IV expression and fibrosis. A lipid-lowering dietary intervention reversed most of these changes. In conclusion, this study indicates that renal fibrosis in early atherosclerosis is a result of a simultaneous increase in extracellular matrix deposition and blunted matrix metalloproteinase–mediated degradation, overall promoting perivascular and tubulointerstitial fibrosis. Notably, many of these pathways may be reversible in hypercholesterolemia, and crucial targets could potentially be identified for early interventions to preserve the kidney.

Objective— Atherosclerotic renovascular disease (ARVD) aggravates renal scarring more than other causes of renal artery stenosis (RAS), but the underlying pathogenic mechanisms of this potential profibrotic effect remain unclear. We tested the hypothesis that coexistence of atherosclerosis and RAS interferes with renal tissue remodeling. Methods and Results— Single-kidney hemodynamics and function were quantified in vivo with electron-beam computed tomography in 3 groups of pigs (n=7 each): normal pigs, pigs 12 weeks after induction of unilateral RAS (RAS group), and pigs with similar-degree RAS fed a 12-week 2% hypercholesterolemic diet (HC+RAS, simulating early ARVD). Kidneys were studied ex vivo by Western blotting and immunohistochemistry. Renal volume, renal blood flow, and glomerular filtration rate were similarly decreased in RAS and HC+RAS ischemic kidneys, accompanied by similar increased expression of profibrotic factors like transforming growth factor-β, tissue inhibitor of metalloproteinase-1, and plasminogen activator inhibitor-1. Nevertheless, HC+RAS kidneys showed increased intrarenal fibrosis compared with RAS-only kidneys. Furthermore, expression of nuclear factor-κB was increased, expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems was decreased, and apoptosis was blunted. Conclusions— Diet-induced HC superimposed on RAS accelerates the development of fibrosis in the stenotic kidney by amplifying profibrotic mechanisms and disrupting tissue remodeling. These alterations might contribute to renal disease progression in ARVD and might account for the increased propensity for end-stage renal disease.

Background— Atherosclerotic renovascular disease may augment deterioration of renal function and ischemic nephropathy compared with other causes of renal artery stenosis (RAS), but the underlying mechanisms remain unclear. This study was designed to test the hypothesis that concurrent early atherosclerosis and hypoperfusion might have greater early deleterious effects on the function and structure of the stenotic kidney. Methods and Results— Regional renal hemodynamics and function at baseline and during vasoactive challenge (acetylcholine or sodium nitroprusside) were quantified in vivo in pigs by electron-beam computed tomography after a 12-week normal (n=7) or hypercholesterolemic (HC, n=7) diet, RAS (n=6), or concurrent HC and a similar degree of RAS (HC+RAS, n=7). Flash-frozen renal tissue was studied ex vivo. Basal cortical perfusion and single-kidney glomerular filtration rate (GFR) were decreased similarly in the stenotic RAS and HC+RAS kidneys, but tubular fluid reabsorption was markedly impaired only in HC+RAS. Perfusion responses to challenge were similarly blunted in the experimental groups. Stimulated GFR increased in normal, HC, and RAS (38.3±3.6%, 36.4±7.6%, and 60.4±9.3%, respectively, P <0.05), but not in HC+RAS (6.5±15.1%). These functional abnormalities in HC+RAS were accompanied by augmented perivascular, tubulointerstitial, and glomerular fibrosclerosis, inflammation, systemic and tissue oxidative stress, and tubular expression of nuclear factor-κB and inducible nitric oxide synthase. Conclusions— Early chronic HC+RAS imposes distinct detrimental effects on renal function and structure in vivo and in vitro, evident primarily in the tubular and glomerular compartments. Increased oxidative stress may be involved in the proinflammatory and progrowth changes observed in the stenotic HC+RAS kidney, which might potentially facilitate the clinically observed progression to end-stage renal disease.