PMC

Gene array analysis reveals sFRP family involvement in hypoxic adaptation of MSC. Total RNA was isolated from MSC and from MSC-HIF and cDNA was synthesized as described in the Methods section. (A) Graphic representation of the array gene expression levels (thresholds in dotted lines). (B) Representative values of the sFRP1-4 and Wnt family gene expression. Values between −3.00 and 3.00 are not considered significant.

sFRP family members possess putative Hypoxia Responsive Elements within their promoter regions. (A) Histogram of the sFRP-1, -3, and -4 genes downregulation and the sFRP-2 gene upregulation. (B) Sequences of the putative HRE found within the promoter regions of the four sFRP genes. NCBI sources : sFRP-1, NC_000074.5, chromosome 8; sFRP-2, NC_000069.5, chromosome 3; sFRP-3, NC_000068.6, chromosome 2; sFRP-4, NC_000079.5, chromosome 13. (C) qRT-PCR validation of sFRP gene expression levels.

HIF-1α overexpression dowregulates ciliogenesis. MSC stably expressing a ΔODD HIF-1α mutant (MSC-HIF) were generated as described in the Methods section. (A) Cells were then cultured under normoxic conditions for 24 hours and cilia staining performed. (B) Quantification of ciliogenesis in MSC (open bars) or MSC-HIF (black bars) was performed. Data are representative of three independent cell cultures. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance between MSC and MSC-HIF.

Schematic summary of the hypoxia-mediated regulation of ciliogenesis in MSC. Hypoxic conditions trigger HIF-1α nuclear translocation which mediates transcriptional downregulation (−) of sFRP-1, -3, -4, and transcriptional upregulation (+) of sFRP-2. These molecular partners lead to inhibition of Wnt signaling and contribute to decreased ciliogenesis. Collectively, the primary cilium may be considered as a differentiation biomarker of cells that must adapt to low oxygen tension microenvironments. Such hypoxic conditions can be found within pathophysiological conditions such as those encountered in hypoxic tumour development, or in physiological regeneration of ischemic tissues.

Overexpression of sFRP-2 or gene silencing of sFRP-1, -3, -4 family members dowregulates ciliogenesis. (A) Gene silencing was performed using specific siRNA for each of the sFRP-1, -2, -3, or -4 members as described in the Methods section. Total RNA was extracted and qRT-PCR performed in order to evaluate efficiency and validate gene expression. (B) Cells were transfected with Mock or sFRP-2 cDNA plasmid, and sFRP-2 gene expression validated by qRT-PCR. (C) Primary cilium staining was performed in Mock- (white bars) and in sFRP-2- (black bars) transfected cells. (D) Gene silencing was performed with a scrambled sequence (siScr; Mock), or with the respective sFRP-1, -2, -3, -4 siRNA sequences. Primary cilium staining was performed in Mock- (white bars) and in si-sFRP-transfected cells (black bars).

Hypoxic culture conditions diminish ciliogenesis in MSC. Mesenchymal stem cells (MSC) were cultured under normoxic or hypoxic conditions as described in the Methods section for 24 and 48 hours. (A) Cells were fixed and cilia staining was performed. (B) Quantification of ciliogenesis in MSC cultured under normoxic (open circles) or hypoxic (black circles) conditions. Data are representative of three independent experiments. Three independent fields were quantified per experiment. Probability values of less than 0.05 were considered significant in hypoxic cultures, and an asterisk (*) identifies such significance relative to the normoxic culture condition.

Hypoxia inducible factor-1α expression is crucial in the hypoxic downregulation of ciliogenesis. MSC were transiently transfected with a scrambled siRNA sequence (Mock) or an siRNA designed to downregulate HIF-1α (siHIF-1α) as described in the Methods section. (A) Cells were then cultured either under normoxic or hypoxic conditions for 24 hours and cilia staining was performed. (B) Quantification of ciliogenesis in MSC cultured under normoxic (open bars) or hypoxic (black bars) conditions. Data are representative of three independent experiments. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance relative to the respective control treatment.