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- Ekman, Mari, et al.
(author)
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HIF-mediated metabolic switching in bladder outlet obstruction mitigates the relaxing effect of mitochondrial inhibition.
- 2014
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In: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 94:5, s. 557-568
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Journal article (peer-reviewed)abstract
- Prior work demonstrated increased levels of hypoxia-inducible factor-1α (HIF-1α) in the bladder following outlet obstruction, associated with bladder growth and fibrosis. Here we hypothesized that HIF induction in outlet obstruction also switches energetic support of contraction from mitochondrial respiration to glycolysis. To address this hypothesis, we created infravesical outlet obstruction in female Sprague-Dawley rats and examined HIF induction and transcriptional activation. HIF-1α increased after 6 weeks of outlet obstruction as assessed by western blotting and yet transcription factor-binding site analysis indicated HIF activation already at 10 days of obstruction. Accumulation HIF-2α and of Arnt2 proteins were found at 10 days, providing an explanation for the lack of correlation between HIF-1α protein and transcriptional activation. HIF signature targets, including Slc2a1, Tpi1, Eno1 and Ldha increased in obstructed compared with sham-operated bladders. The autophagy markers Bnip3 and LC3B-II were also increased at 6 week of obstruction, but electron microscopy did not support mitophagy. Mitochondria were, however, remodeled with increased expression of Cox4 compared with other markers. In keeping with a switch toward glycolytic support of contraction, we found that relaxation by the mitochondrial inhibitor cyanide was reduced in obstructed bladders. This was mimicked by organ culture with the HIF-inducer dimethyloxalylglycine, which also upregulated expression of Ldha. On the basis of these findings, we conclude that HIF activation in outlet obstruction involves mechanisms beyond the accumulation of HIF-1α protein and that it results in a switch of the energetic support of contraction to anaerobic glycolysis. This metabolic adaptation encompasses increased expression of glucose transporters and glycolytic enzymes combined with mitochondrial remodeling. Together, these changes uphold contractility when mitochondrial respiration is limited.Laboratory Investigation advance online publication, 3 March 2014; doi:10.1038/labinvest.2014.48.
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- Fitchev, Philip P, et al.
(author)
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Thrombospondin-1 regulates the normal prostate in vivo through angiogenesis and TGF-beta activation
- 2010
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In: Laboratory Investigation. - : Elsevier BV. - 0023-6837 .- 1530-0307. ; 90:7, s. 1078-1090
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Journal article (peer-reviewed)abstract
- Castration experiments in rodents show that the stromal vasculature is critical to the androgen-mediated prostate growth regulation. However, the role of angiogenesis inhibitors, such as thrombospondin-1 (TSP-1), in this process is unclear. TSP-1 is a multifunctional glycoprotein that can function as a potent angiogenesis inhibitor and an in vivo activator of latent transforming growth factor-beta (TGF-beta) in some tissues. On the basis of these observations, we hypothesized that TSP-1 regulated androgen withdrawal-induced prostate regression and that this process was mediated not only through antiangiogenic activity but also through TGF-beta activation. To test this, we evaluated angiogenic activity in human prostate epithelial and stromal cells treated with androgens and hypoxia in vitro. TSP-1 knockout mice were characterized to investigate the in vivo functions of TSP-1. In vitro, we found that androgens and hypoxia differentially regulated TSP-1 and angiogenic activity. Androgens stimulated normal epithelial cell, but inhibited normal stromal cell, angiogenic activity. Conversely, hypoxia stimulated stromal while inhibiting epithelial activity. Thus, in vivo, net angiogenic activity must reflect cellular interactions. And, we found that media conditioned by epithelial cells grown under normoxic conditions stimulated stromal cell angiogenic activity, and if epithelial cells were grown under hypoxic conditions, stromal activity was further increased. TSP-1 levels, however, were unchanged. In vivo, TSP-1 loss in a mouse model led to prostate epithelial hyperplasia by 3 months of age with only a modest stromal effect. Androgens suppressed TSP-1 as expression increased after castration both in normal mouse prostate and in human prostate cancer tissues. In addition, TSP-1 expression corresponded to increased TGF-beta activation in mouse tissues, specifically in the stromal compartment. These data show a critical role for TSP-1 in prostate epithelial and stromal growth regulation through angiogenic inhibition and activation of latent TGF-beta. Therefore, loss of TSP-1 during tumorigenesis would eliminate two barriers to cancer progression.
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- Hwaiz, Rundk, et al.
(author)
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Rac1 regulates platelet shedding of CD40L in abdominal sepsis.
- 2014
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In: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 94:9, s. 1054-1063
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Journal article (peer-reviewed)abstract
- Matrix metalloproteinase-9 (MMP-9) regulates platelet shedding of CD40L in abdominal sepsis. However, the signaling mechanisms controlling sepsis-induced shedding of CD40L from activated platelets remain elusive. Rac1 has been reported to regulate diverse functions in platelets; we hypothesized herein that Rac1 might regulate platelet shedding of CD40L in sepsis. The specific Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6-methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride) was administered to mice undergoing cecal ligation and puncture (CLP). Levels of CD40L and MMP-9 in plasma, platelets, and neutrophils were determined by use of ELISA, western blot, and confocal microscopy. Platelet depletion abolished the CLP-induced increase in plasma levels of CD40L. Rac1 activity was significantly increased in platelets from septic animals. Administration of NSC23766 abolished the CLP-induced enhancement of soluble CD40L levels in the plasma. Moreover, Rac1 inhibition completely inhibited proteinase-activated receptor-4-induced surface mobilization and secretion of CD40L in isolated platelets. CLP significantly increased plasma levels of MMP-9 and Rac1 activity in neutrophils. Treatment with NSC23766 markedly attenuated MMP-9 levels in the plasma from septic mice. In addition, Rac1 inhibition abolished chemokine-induced secretion of MMP-9 from isolated neutrophils. Finally, platelet shedding of CD40L was significantly reduced in response to stimulation with supernatants from activated MMP-9-deficient neutrophils compared with supernatants from wild-type neutrophils, indicating a direct role of neutrophil-derived MMP-9 in regulating platelet shedding of CD40L. Our novel data suggest that sepsis-induced platelet shedding of CD40L is dependent on Rac1 signaling. Rac1 controls surface mobilization of CD40L on activated platelets and MMP-9 secretion from neutrophils. Thus, our findings indicate that targeting Rac1 signaling might be a useful way to control pathologic elevations of CD40L in the systemic circulation in abdominal sepsis.Laboratory Investigation advance online publication, 21 July 2014; doi:10.1038/labinvest.2014.92.
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- Macchia, Gemma, et al.
(author)
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FOSL1 as a candidate target gene for 11q12 rearrangements in desmoplastic fibroblastoma.
- 2012
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In: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 92:5, s. 735-743
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Journal article (peer-reviewed)abstract
- Desmoplastic fibroblastoma (DF) is a benign fibroblastic/myofibroblastic tumor. Cytogenetic analyses have revealed consistent rearrangement of chromosome band 11q12, strongly suggesting that this region harbors a gene of pathogenetic importance. To identify the target gene of the 11q12 rearrangements, we analyzed six cases diagnosed as DF using chromosome banding, fluorescence in situ hybridization (FISH), single-nucleotide polymorphism array and gene expression approaches. Different structural rearrangements involving 11q12 were found in five of the six cases. Metaphase FISH analyses in two of them mapped the 11q12 breakpoints to an ∼20-kb region, harboring FOSL1. Global gene expression profiling followed by quantitative real-time PCR showed that FOSL1 was expressed at higher levels in DF with 11q12 rearrangements than in desmoid-type fibromatoses. Furthermore, FOSL1 was not upregulated in the single case of DF that did not show cytogenetic involvement of 11q12; instead this tumor was found to display a hemizygous loss on 5q, including the APC (adenomatous polyposis coli) locus, raising the possibility that it actually was a misdiagnosed Gardner fibroma. 5'RACE-PCR in two 11q12-positive DF did not identify any fusion transcripts. Thus, in agreement with the finding at chromosome banding analysis that varying translocation partners are involved in the 11q12 rearrangement, the molecular data suggest that the functional outcome of the 11q12 rearrangements is deregulated expression of FOSL1.Laboratory Investigation advance online publication, 12 March 2012; doi:10.1038/labinvest.2012.46.
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