Interleukin-1β sequesters hypoxia inducible factor 2α to the primary cilium.

BACKGROUND: The primary cilium coordinates signalling in development, health and disease. Previously we have shown that the cilium is essential for the anabolic response to loading and the inflammatory response to interleukin-1β (IL-1β). We have also shown the primary cilium elongates in response to IL-1β exposure. Both anabolic phenotype and inflammatory pathology are proposed to be dependent on hypoxia-inducible factor 2 alpha (HIF-2α). The present study tests the hypothesis that an association exists between the primary cilium and HIFs in inflammatory signalling. RESULTS: Here we show, in articular chondrocytes, that IL-1β-induces primary cilia elongation with alterations to cilia trafficking of arl13b. This elongation is associated with a transient increase in HIF-2α expression and accumulation in the primary cilium. Prolyl hydroxylase inhibition results in primary cilia elongation also associated with accumulation of HIF-2α in the ciliary base and axoneme. This recruitment and the associated cilia elongation is not inhibited by blockade of HIFα transcription activity or rescue of basal HIF-2α expression. Hypomorphic mutation to intraflagellar transport protein IFT88 results in limited ciliogenesis. This is associated with increased HIF-2α expression and inhibited response to prolyl hydroxylase inhibition. CONCLUSIONS: These findings suggest that ciliary sequestration of HIF-2α provides negative regulation of HIF-2α expression and potentially activity. This study indicates, for the first time, that the primary cilium regulates HIF signalling during inflammation

The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKγ/NF-κB-dependent pathway

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP’s catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells

A Concerted HIF-1α/MT1-MMP Signalling Axis Regulates the Expression of the 3BP2 Adaptor Protein in Hypoxic Mesenchymal Stromal Cells

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour

The Primary Cilium as a Biomarker in the Hypoxic Adaptation of Bone Marrow-Derived Mesenchymal Stromal Cells: A Role for the Secreted Frizzled-Related Proteins

A pivotal role in guiding mesenchymal stem cell (MSC) differentiation has recently been attributed to the primary cilium. This solitary, non-motile microtubule-based organelle emerging from the cell surface acts as a sensorial membrane structure reflecting developmental and adaptive processes associated with pathologies including human cystic kidney disease, skeletal malformations, obesity and cancer. Given that the intrinsic hypoxic adaptation of MSC remains poorly understood within ischemic tissues or hypoxic tumours, we questioned whether the hypoxia inducible factor-1α (HIF-1α) might be a downstream effector regulating cilium maintenance. We show that murine bone marrow-derived MSC cultured under hypoxic conditions (1.2% O 2 ) lose their primary cilia in a time-dependent manner. Gene silencing of HIF-1α prevented cilia loss in hypoxic cultures, and generation of MSC expressing a constitutively active HIF-1α (MSC-HIF) was found to decrease primary cilium formation. A Wnt pathway-related gene expression array was also performed on MSC-HIF and indicated that the secreted Frizzled-related proteins (sFRP)-1, –3 and –4 were down-regulated, while sFRP-2 was up-regulated. Overexpression of recombinant sFRP-2 or gene silencing of sFRP-1, –3 and –4 in MSC led to primary cilium disruption. These results indicate a molecular signalling mechanism for the hypoxic disruption of the primary cilium in MSC involving an HIF-1α/sFRP axis. This mechanism contributes to our understanding of the adaptive processes possibly involved in the oncogenic transformation and tumour-supporting potential of MSC. Our current observations also open up the possibility for the primary cilia to serve as a biomarker in MSC adaptation to low oxygen tension within (patho)physiological microenvironments

Abstract 4027: The lectin concanavalin-A induces cyclooxygenase-2 expression through an IKKgamma/NFkB-dependent pathway in U87 glioblastoma cells

Abstract The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) and is, therefore, routinely used to assess MT1-MMP-mediated functions. Given that gliomas with increasing histological grade demonstrated MT1-MMP expression that correlated with high expression of cyclooxygenase (COX)-2, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA, a lectin known to induce MT1-MMP expression, in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA and MT1-MMP-mediated intracellular signaling and induction of COX-2 was also validated in both wild-type and Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2 by ConA. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4027.</jats:p

Gene silencing of MT1-MMP antagonizes the effects of hypoxia on 3BP2 gene expression.

<p>MSC were transiently transfected with scrambled sequences (Mock, white bars) or MT1-MMP siRNA (black bars) as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions. (A) Immunoblotting of MT1-MMP protein expression was evaluated in cell lysates from siRNA experiments. Total RNA was extracted, and qRT-PCR was used to assess 3BP2, HIF-1α and MT1-MMP. Apoptotic cell death was assessed using the fluorometric caspase-3 activity assay as described in the Methods section. (B) 3BP2 and HIF-1α gene expression were assessed by qRT-PCR in Mock-transfected (open circles) and in siMT1-MMP-transfected (closed circles) cells that were subsequently cultured under hypoxic conditions. (C) MSC were transiently transfected with either scrambled sequences or MT1-MMP siRNA as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions, cell lysates were isolated, western blotting and immunodetection were performed with anti-3BP2 and anti-GAPDH antibodies. Values in (A) and (B) are means of two independent experiments, each performed in triplicates (*<i>p</i><0.05 versus mock control in (A) or mock at time = 0 hr hypoxia in (B)); Bars, ±SD.</p

Hypoxic culture conditions upregulate 3BP2 gene expression.

<p>Subconfluent MSC were serum-starved and cultured under normoxic (5% CO₂ and 95% air; white box) or hypoxic (1% O₂, 5% CO₂ and 94% N₂; black box) conditions for 18 hours. Total RNA was extracted and quantitative reverse transcription-polymerase chain reaction was performed in order to assess 3BP2, HIF-1α, VEGF and MT1-MMP gene expression levels. Values are means of two independent experiments, each performed in triplicates. Bars, ±SD.</p