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

3BP2 induction requires MT1-MMP cytoplasmic domain-mediated signalling.

<p>(A) MSC were transiently transfected with pcDNA3.1 (Mock, white bars), a plasmid encoding full length MT1-MMP (Wt, black bars), or a plasmid encoding for a cytoplasmic domain-deleted form of MT1-MMP (Δ-cyto, grey bars). Cells were then cultured under normoxic culture conditions, total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression. (B) Gelatin zymography of the conditioned media was used to demonstrate efficient cell surface targeting of the respective Wt and Δ-cyto MT1-MMP recombinant proteins and subsequent ability to retain extracellular catalytic functions and to activate the secreted latent proMMP-2 into active MMP-2. (C) Cell lysates were isolated, western blotting and immunodetection was performed with antibodies recognizing the MT1-MMP catalytic or cytoplasmic domain, anti-3BP2 and anti-GAPDH as described in the Methods section. Values are means of two independent experiments, each performed in triplicates (*<i>p</i><0.05 versus Wt-MT1-MMP control); Bars, ±SD.</p

Gene silencing of HIF-1α antagonizes the effects of hypoxia on 3BP2 gene and protein expression.

<p>MSC were transiently transfected with scrambled sequences (Mock, white bars, open circles) or HIF-1α siRNA (black bars, closed circles) as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021511#pone-0021511-g001" target="_blank">Fig. 1</a>. (A) Apoptotic cell death was assessed using the fluorometric caspase-3 activity assay as described in the Methods section. Concanavalin-A-treated MSC (grey bars) was used as a positive inducer of caspase-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021511#pone.0021511-Annabi3" target="_blank">[16]</a>. Total RNA was extracted, and qRT PCR was used to assess 3BP2 and HIF-1α transcript levels. (B) 3BP2 gene expression was assessed by qRT-PCR in Mock-transfected and in siHIF-1α-transfected cells that were subsequently cultured under hypoxic conditions. (C) Mesenchymal stromal cells were transiently transfected with scrambled sequences or HIF-1α 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 was performed with anti-3BP2 and anti-GAPDH antibodies. A representative blot is shown out of two. (D) Scanning densitometry was used to assess protein expression described in panel C, and the ratio of 3BP2/GAPDH expression was represented. 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

Stable expression of HIF-1α increases 3BP2 basal expression and MSC migration.

<p>MSC stably expressing a ΔODD HIF-1α mutant (MSC-HIF) were generated as described in the Methods section. (A) Basal migration of MSC and MSC-HIF was performed as described in the Methods section. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic MSC). (B) Cell lysates were isolated, western blotting and immunodetection was performed with anti-3BP2 and anti-GAPDH antibodies as described in the Methods section. (C) Total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression. (D) Cell migration was quantified as described in the Methods section in Mock- (white bars) and si3BP2- (black bars) transfected MSC or MSC-HIF-1α. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic mock MSC or MSC-HIF); Bars, ±SD.</p

Putative mechanisms involved in 3BP2 response to hypoxia and recruitment by MT1-MMP.

<p>Scheme summarizing the mechanisms involved in 3BP2 response to hypoxia and potential recruitment by MT1-MMP. Hypoxia induces HIF-1α, which then upregulates MT1-MMP gene transcription, MT1-MMP synthesis and targeting to the plasma membrane. The intracellular domain of MT1-MMP is mandatory to signal potential 3BP2 recruitment and to trigger both cell migration and extracellular matrix (ECM) degradation. proMMP-2, latent form of matrix metalloproteinase-2; MMP-2, active form of matrix metalloproteinase-2; TIMP-2; tissue inhibitor of MMP-2.</p