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

Rôle de la protéine adaptatrice 3BP2/SH3BP2 dans la régulation de l'homéostasie osseuse

Les ostéoclastes sont des cellules multinucléées capables de résorber le tissu osseux, elles se différencient à partir de la lignée hématopoïétique en présence de Receptor Activator of Nuclear Factor NF-kB (RANK-L) et M-CSF (Macrophage-Colony Stimulating Factor). Les récepteurs RANK et M-CSFR relaient et amplifient le signal de leurs ligands en activant de multiples voies de signalisation intracellulaire dont l intégration aboutira à l intégration de NFATc1, facteur de transcription essentiel pour la différenciation des ostéoclastes. Les protéines adaptatrices, du fait de leur structure, jouent un rôle crucial dans cette signalisation. De nombreuses études ont montré que la protéine adaptatrice 3BP2/ SH3BP2, initialement identifiée comme une protéine interagissant avec la kinase c-Abl puis comme partenaire des kinases de la famille Src et Syk, joue un rôle important dans la signalisation et l activation des leucocytes. Des études génétiques ont montré que des mutations du gène 3bp2 chez l homme sont associées à une dysplasie osseuse génétique Chérubinisme et à un phénotype ostéopénique inflammatoire chez la souris. Ces observations laissent entrevoir un rôle additionnel de 3BP2 dans la régulation des cellules du système osseux et particulièrement la différenciation des ostéoclastes. Dans le but d étudier le rôle de 3BP2 dans la différenciation des ostéoclastes, nous avons utilisé la lignée RAW264.7, une lignée myélo-monocytaire murine capable de se différencier en ostéoclaste en présence de RANK-L. Avec la méthode d interférence ARN, nous avons développé des modèles cellulaires perte de fonction n exprimant plus 3BP2. Ces modèles nous ont permis de montrer que l expression de 3BP2 est essentielle pour la différenciation des ostéoclastes et que son absence altère la différenciation de ces cellules en ostéoclastes. Nous avons ainsi montré que l effet de l absence de 3BP2 est restreint à la voie de signalisation RANK sans aucune conséquence sur la voie de signalisation GM-CSF et la différenciation des cellules dendritiques. Dans un premier temps, nous avons montré que cet effet perte de fonction est lié à un défaut de polymérisation d actine et d activation de la protéine tyrosine kinase Src et des voies de signalisation MAPKs (MEK, ERK, JNK) contrôlées par RANK-L. Nous avons observé également une inhibition de l induction de NFATc1 et AP-1, des facteurs de transcription essentiels à la différenciation des ostéoclastes. Par la suite, l analyse transcriptionnelle de différentes protéines impliquées dans la différenciation des ostéoclastes (CaR, TRAP, Cathépsine K) a révélée une inhibition significative de leur induction dans les cellules déficients en 3BP2. Nous avons finalement montré que la reconstruction avec des molécules Src et NFATc1 constitutivement actives restaure la différenciation des ostéoclastes dans les cellules déficients en 3BP2. En conclusion, notre étude a montré que la protéine 3BP2, via la protéine tyrosine kinase Src, joue un rôle central au cours de la différenciation des ostéoclastes en contrôlant les voies de signalisation RANK, impliquées dans l activation de NFATc1, facteur de transcription clé de la différenciation ostéoclastique.Osteoclasts are multinucleated bone-resorbing cells, which derived from hematopoietic cells of the monocyte/macrophage lineage following stimulation with two essential cytokines, RANK-L and M-CSF. The molecular pathways involved in osteoclast formation involve complex network of signaling molecules, including adaptor proteins kinases, which ultimately lead to the activation of a transcriptional program in which NFATc1 plays a pivotal role. The adaptor protein 3BP2, originally identified as a c-Abl binding protein, and a partner of Src and Syk kinases families, has been involved in leucocytes signaling and activation? Genetic studies have further associated mutations of the 3BP2 gene of the human bone disease Cherubism and to inflammation and bone dysfunction in mouse. However, how wild-type 3BP2 exactly functions in osteoclast differentiation has yet been elucidated. In this study, we have investigated the role of endogenous 3BP2 exactly functions in osteoclast differentiation using siRNA-mediated silencing of 3BP2 expression in the RAW264.7 monocyte/macrophage cell line. We show here that 3BP2 was required for RANK-L-induced differentiation of RAW264.7 cells was associated with reduced RANK-L-induced actin reorganization and Src, ERK, JNK, IKKa/b, but not p38 phosphorylation. Following RANK-L stimulation, the 3BP2-deficient cells exhibited impaired up-regulation of Src, c-fos and NFATC1 mRNA expression, whereas NFATc2 and NFATc3messengers were not significantly affected. Compared to control cells, 3BP2-knockdown cells induced to osteoclast by RANK-L displayed no up-regulation of Src and NFATc1 proteins? In addition, the introduction of constitutively active mutants of Src and NAFTc1 in 3NP2 deficient cells restored osteoclast differentiation. Finally, we provide evidence that enhanced osteoclast differentiation triggered by a 3BP2 Cherubism mutant also required NFAT activity in RAW264.7 cells. Together, this study demonstrates that 3BP2 is a key regulator of RANK-mediated osteoclastogenesis through Src and NFATc1 activation.NICE-BU Sciences (060882101) / SudocSudocFranceF

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

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

3BP2 silencing abrogates MSC migration in response to hypoxic culture conditions.

<p>MSC were transiently transfected with scrambled sequences or with 3BP2 siRNA as described in the Methods section. (A) Immunoblotting of 3BP2 protein expression was evaluated in cell lysates from Mock (white bars) and siRNA (black bars) experiments. Total RNA was extracted, and qRT-PCR was used to assess 3BP2 gene expression knockdown efficiency. Apoptotic cell death was assessed by the fluorometric caspase-3 activity as described in the Methods section. (B) Cells were harvested and seeded onto Boyden chambers using gelatin-coated filters to assess cell migration under normoxic or hypoxic culture conditions. (C) Cell migration was quantified as described in the Methods section and expressed as x-fold induction over Mock-transfected cells. Values of cell migration are means of three independent experiments (*<i>p</i><0.05 versus normoxic mock); 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

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

Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway

AbstractThe small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation