Squamous cell cancers account for more than half of all human cancers, and esophageal cancer is the sixth leading cause of cancer death worldwide. The majority of esophageal squamous cell carcinomas have identifiable p53 mutations, yet the same p53 mutations are found at comparable frequencies in pre-cancerous dysplasia, indicating that transformation requires additional somatic changes yet to be defined. Here we show that the zinc finger transcription factor KLF5 transactivates NOTCH1 in the context of p53 mutation or loss. KLF5 loss limited NOTCH1 activity and was sufficient on its own to transform primary human keratinocytes harboring mutant p53, leading to formation of invasive tumors. Restoration of NOTCH1 blocked transformation of KLF5-deficient and p53 mutant keratinocytes. While human dysplastic epithelia accumulated KLF5, KLF5 expression was lost concurrently with NOTCH1 in squamous cell cancers. Taken together, these results define KLF5 loss as a critical event in squamous cell transformation and invasion. Our findings suggest that KLF5 may be a useful diagnostic and therapeutic target in esophageal squamous carcinomas and possibly more generally in other cancers associated with p53 loss-of-function.
Looft-Wilson RC, Ashley BS, Billig JE, Wolfert MR, Ambrecht LA, Bearden SE. Chronic diet-induced hyperhomocysteinemia impairs eNOS regulation in mouse mesenteric arteries. Am J Physiol Regul Integr Comp Physiol 295: R59 -R66, 2008. First published April 30, 2008 doi:10.1152/ajpregu.00833.2007.-Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO ⅐ ) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO ⅐ , endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO ⅐ and non-NO ⅐ vasodilatory mechanisms, undergo alteration of non-NO ⅐ mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hcy: 89.2 Ϯ 49.0 M) compared with age-matched controls (Hcy: 6.6 Ϯ 1.9 M), using first-order mesenteric arteries. Dilation to ACh (10 Ϫ9 -10 Ϫ4 M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without N G -nitro-L-arginine methyl ester (L-NAME) (10 Ϫ4 M) and/or indomethacin (10 Ϫ5 M) to test endothelium-dependent dilation and non-NO ⅐ -dependent dilation, respectively. The time course of dilation to ACh (10 Ϫ4 M) was examined to compare the initial transient dilation due to non-NO ⅐ , non-prostacyclin mechanism and the sustained dilation due to NO ⅐ . These experiments indicated that endothelium-dependent dilation was attenuated (P Ͻ 0.05) in HHcy arteries due to downregulation of only NO ⅐ -dependent dilation. Western blot analysis indicated significantly less (P Ͻ 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic dietinduced HHcy in mice impairs eNOS protein expression and phosphorylation at S1179, coincident with impaired NO ⅐ -dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy.
<b><i>Background:</i></b> Arteries chronically constricted in culture remodel to smaller diameters. Conversely, elevated luminal shear stress (SS) promotes outward remodeling of arteries in vivo and prevents inward remodeling in culture in a nitric oxide synthase (NOS)-dependent manner. <b><i>Objectives:</i></b> To determine whether SS-induced prevention of inward remodeling in cultured arteries is specifically eNOS-dependent and requires dilation, and whether SS alters the expression of eNOS and other genes potentially involved in remodeling. <b><i>Methods:</i></b> Female mouse thoracodorsal arteries were cannulated, pressurized to 80 mm Hg, and cultured for 2 days with low SS (<7 dyn/cm<sup>2</sup>), high SS (≥15 dyn/cm<sup>2</sup>), high SS + L-NAME (NOS inhibitor, 10<sup>–4</sup> M), or high SS in arteries from eNOS–/– mice. In separate arteries cultured 1 day with low or high SS, eNOS and connexin (Cx) 37, Cx40, and Cx43 mRNA were assessed with real-time PCR. <b><i>Results:</i></b> High SS caused little change in passive diameters after culture (–4.7 ± 2.0%), which was less than low SS (–18.9 ± 1.4%; <i>p</i> < 0.0001), high SS eNOS–/– (–18.0 ± 1.5; <i>p</i> < 0.001), or high SS + L-NAME (–12.0 ± 0.6%; nonsignificant) despite similar constriction during culture. Cx37 mRNA expression was increased (<i>p</i> < 0.05) with high SS, but other gene levels were not different. <b><i>Conclusions:</i></b> eNOS is involved in SS-induced prevention of inward remodeling in cultured small arteries. This effect does not require NO-mediated dilation. SS increased Cx37.
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