Hypoxia and Hypoxia Mimetics Decrease Aquaporin 5 (AQP5) Expression through Both Hypoxia Inducible Factor-1α and Proteasome-Mediated Pathways

The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.</p

Aerosol Gemcitabine after Amputation Inhibits Osteosarcoma Lung Metastases but Not Wound Healing

Background. In newly diagnosed osteosarcoma (OS) patients, the time between surgery and resumption of chemotherapy is 2–7 weeks. Delays \u3e 16 days are associated with increased risk of relapse and decreased overall survival. Identifying an e4ective therapy that can be used postoperatively may prevent relapse. We investigated whether aerosol gemcitabine (GCB) initiated after tumor resection inhibited the growth of OS lung metastases without a4ecting the wound-healing process. Methods. Mice were injected intratibially with OS cells. Amputation was performed when the tumor reached 1.5 cm. Fullthickness excisional wounds were also made on the dorsal skin and tail. Aerosol GCB or PBS was initiated 48 hours after amputation (3 times/week for 3 weeks). Wound sections were evaluated by immunohistochemistry for Ki-67 (proliferation), CD31 (vessels), VEGF, IL-10, bFGF, mast cells, macrophages, and M1/M2 macrophage ratios. )e lungs were analyzed for macro- and micrometastases. Results. Aerosol GCB inhibited the growth of the lung metastases but had no e4ect on the 3 phases of wound healing in the dorsal skin, tail, or bone. Production of cytokines at the wound sites was the same. Conclusion. )ese data indicate that initiating aerosol GCB postoperatively may kill residual lung metastases thereby preventing relapse and improve survival

Aerosol Gemcitabine after Amputation Inhibits Osteosarcoma Lung Metastases but Not Wound Healing

Background. In newly diagnosed osteosarcoma (OS) patients, the time between surgery and resumption of chemotherapy is 2–7 weeks. Delays > 16 days are associated with increased risk of relapse and decreased overall survival. Identifying an effective therapy that can be used postoperatively may prevent relapse. We investigated whether aerosol gemcitabine (GCB) initiated after tumor resection inhibited the growth of OS lung metastases without affecting the wound-healing process. Methods. Mice were injected intratibially with OS cells. Amputation was performed when the tumor reached 1.5 cm. Full-thickness excisional wounds were also made on the dorsal skin and tail. Aerosol GCB or PBS was initiated 48 hours after amputation (3 times/week for 3 weeks). Wound sections were evaluated by immunohistochemistry for Ki-67 (proliferation), CD31 (vessels), VEGF, IL-10, bFGF, mast cells, macrophages, and M1/M2 macrophage ratios. The lungs were analyzed for macro- and micrometastases. Results. Aerosol GCB inhibited the growth of the lung metastases but had no effect on the 3 phases of wound healing in the dorsal skin, tail, or bone. Production of cytokines at the wound sites was the same. Conclusion. These data indicate that initiating aerosol GCB postoperatively may kill residual lung metastases thereby preventing relapse and improve survival

Statistiques des relations de travail

<p>Expression of Proteasome subunit encoding genes, that were significantly changed in the presence of hypoxic stress induced by Co⁺⁺. Cells were treated either with vehicle or Co⁺⁺ (n = 3 independent biological samples, n = 3 microarray chips per biological sample). Microarray analysis was performed to identify differentially expressed genes after addition of Co⁺⁺, using R statistical software and the <i>limma</i> Bioconductor package.</p

Hypoxia and Cobalt regulate expression of AQP5 via a proteasome dependent pathway.

<p>(A) Western blot analysis to determine AQP5 expression in total protein extracts isolated from cells exposed to normoxia, hypoxia or hypoxia with 10 μm proteasome inhibitor PI III for 24 h. β-actin is used as a loading control. (B)& (C) Western blot analysis of total protein extracts of cells were pre-treated with 10 μM proteasome inhibitor PI III or LC for 30 min, and then 100 μM Cobalt was added for 24 h. β-actin is used as loading control. (D) Quantitation of Western blots in panels A–C. (E)& (F) Northern blot analysis of total mRNA isolated from cells, which were pre-treated with 10 μM proteasome inhibitor PI III or LC for 30 min, and then 100 μM cobalt was added for 24 h. L32 is used as a loading control. (G) Quantitation of Northern blots in panels E and F. (H) Western blot analysis to determine HIF-1α expression in nuclear extracts from differently treated cells. Lane 1, normoxia; lane 2, hypoxia; lane 3, normoxia plus cobalt; lane 4, normoxia plus PI III; lane 5, hypoxia plus PI III; lane 6, normoxia plus PI III and cobalt. Values for the blots are the mean ± SD (n = 3).</p

<i>de novo</i> protein synthesis is necessary to down-regulate AQP5 expression.

<p>(A) Western blot analysis of MLE-12 cells pre-incubated with cycloheximide or vehicle (70% ethanol) followed by 100 μM cobalt. (B) Quantitation of the Western blots in panel B using β-actin as the loading control. (C) Northern blot analysis of MLE-12 cells pre-incubated with cycloheximide or vehicle (70% ethanol) followed by 100 μM cobalt. (D) Quantitation of the Northern blots in panel E using L32 as the loading control. Values in the all the blots are the mean ± SD (n = 3).</p

A putative model for the molecular pathway linking hypoxic stress to decreased AQP5 expression.

<p>Hypoxic stress activates HIF-1α via the ERK signaling pathway. HIF-1α activates a repressor of AQP5 transcription via a proteasome-mediated mechanism (which could act by degrading inhibitors of the repressor protein).</p

Cobalt regulates AQP5 expression via ERK signaling pathway.

<p>(A) Western blot analysis to determine AQP5 expression in total protein extracts isolated from cells treated with vehicle, cobalt, cobalt plus ERK pathway inhibitor PD98059 and PD98059 alone. (B) Quantitation of the Western blots in panel A using β-actin as the loading control. (C) Western blot analysis to determine phosphorylated ERK expression (upper panel) in total protein extracts isolated from cells shown in panel A. Total ERK expression in the same groups (lower panel). (D) Quantitation of the change in phosphorylation of ERK using total ERK for normalization. (E) Western blot analysis to determine HIF-1α expression in nuclear protein extracts isolated from cells treated with vehicle, cobalt, cobalt plus ERK pathway inhibitor PD98059 and PD98059 alone. (F) Quantitation of the Western blots in panel E using Lamin B as the loading control. Values for the blots are the mean ± SD (n = 3).</p