Conserved regulation of the Jak/STAT pathway by the endosomal protein asrij maintains stem cell potency

Abstract

Asrij/OCIAD1 is an endosomal protein expressed in stem cells and cardiovascular lineages and aberrantly expressed in several cancers. We show that dose-dependent modulation of cytokine-dependent JAK/STAT signaling by Asrij regulates mouse embryonic stem cell pluripotency as well as Drosophila hematopoietic stem cell maintenance. Furthermore, mouse asrij can substitute for Drosophila asrij, indicating that they are true homologs. We identify a conserved region of Asrij that is necessary and sufficient for vesicular localization and function. We also show that Asrij and STAT3 colocalize in endosomes and interact biochemically. We propose that Asrij provides an endosomal scaffold for STAT3 interaction and activation, and may similarly control other circuits that maintain stemness. Thus, Asrij provides a key point of control for spatial and kinetic regulation of stem cell signals.

Figure 1. Asrij Maintains mESC Self-Renewal and Pluripotency

Analysis of asrij-modulated ESC lines. (A) Expression of mRNA (i) and protein (ii). (B) Morphology. (C) Quantitative analysis of proliferation over 48 hr (i), and population doubling time analysis (i and ii). Panel (ii) shows increased doubling time for +/− cells and faster doubling for OV cells, and panel (iii) shows reduced and increased percentages of dividing cells for +/− and OV cells, respectively. (D) Cell-cycle profiles analyzed by flow cytometry. Bars represent % distribution of cells in G1, S, and G2-M. (E) Image (i) and graph (ii) showing AP+ colonies in a clonal assay. (F) Quantitative RT-PCR (qRT-PCR) analysis for pluripotency markers. Statistical significance is indicated by **p < 0.05, ***p < 0.001. Error bars show SD of the mean. Scale bar, 100 μM. See also Figure S1.

Figure 2. Asrij Reduces the LIF Dependence of ESCs and Promotes STAT3 Phosphorylation

mESC lines were cultured on 0.1% gelatin in media without LIF for 48 hr unless indicated otherwise. In all cases, values for +/− and OV were compared with those for +/+ cells. (A–C) Graph representing (A) cell proliferation over 4 days, (B) clonogenicity, and (C) the effect of serial subculture at clonal density for three passages (p). (D and E) qRT-PCR analysis of (D) pluripotency marker and (E) lineage differentiation marker gene expression. (F–H) Western blot and graphical representation showing (F–H) pSTAT3 level in culture (F) with LIF or (G) after 4 days of LIF withdrawal or (H) upon treatment with a JAK inhibitor. (I) pERK level. Statistical significance is indicated by **p < 0.01, ***p < 0.001. Error bars show SD of the mean. See also Figure S2.

Figure 3. Conserved Role for Asrij in Regulating JAK/STAT Activity and Maintaining Stemness

(A) Schematic representation of the Drosophila primary lymph gland lobe. (B–M) The primary lobes of control and mutant/modulated larval lymph glands were assessed and compared. Genotypes are as indicated. (B) Domeless expression marked by DomelessGFP is lost in the asrij null mutant with precocious differentiation into P1+ plasmatocytes. (C) A JAK/STAT pathway activation reporter assay shows highly increased stat-GFP activation upon asrij overexpression and decreased statGFP activity in the asrij null mutant. The graph shows the average statGFP intensity values for each genotype (n = 10). (D) Increased hemocyte differentiation is seen upon stat92e knockdown in Asrij overexpression larvae. (E) Asrij overexpression in the collier mutant larval lymph gland can rescue premature differentiation into P1+ plasmatocytes and restore a fully functional Antennapedia+ niche. (F and G) Primary lymph gland lobes of unpaired and domeless hypomorphs phenocopy asrij null mutants as seen by P1 expression (F), and have fewer Antennapedia+ niche cells as indicated in the graph (G). (H) Precocious differentiation seen in the asrij null mutant is repressed upon expression of mouse asrij as seen by P1+ plasmatocytes and ProPO+ crystal cells. (I) Schematic representation of Asrij full-length protein showing the N-terminal fragment containing the OCIA domain (ArjN, gray bar) and the C-terminal fragment lacking the domain (ArjC, black bar). Putative hydrophobic stretches are indicated (white bars). (J) HEK293 cells bearing FLAG-tagged Asrij fragments and immunostained to visualize localization of the fragment ArjN or ArjC. (K) Lymph gland lobes of wild-type larvae additionally expressing ArjN or ArjC and stained for P1+ plasmatocytes show premature differentiation in ArjC lymph glands. (L) Premature differentiation in the asrij null mutant can be rescued by forced expression of ArjN, but not ArjC. (M) Reduced dominant-negative effect of ArjC in Asrij overexpressing lobes. Statistical significance is indicated by ***p < 0.001. Nuclei were viewed with DAPI staining and the image was used to draw the lymph gland boundary (white line). Scale bars, 50 μm (B–H and K–M) and 12.5 μm (J).

Figure 4. Overexpression of ArjC Reduces STAT3 Phosphorylation in ESCs

Analysis of arjN and arjC ESC lines. (A) Morphology. (B) Graph showing AP+ colonies in a clonal assay. (C and D) qRT-PCR analysis showing expression of pluripotency markers (C) and differentiation markers (D). (E and F) Western blot and graphical representation showing the (E) pSTAT3 level and (F) pERK level. GAPDH was used as a normalizing control. Statistical significance is indicated by *p < 0.02, **p < 0.05, ***p < 0.001. Error bars show SD of the mean. Scale bar, 10 μM. See also Figure S3.

Figure 5. Asrij Interacts with STAT3

(A) HEK293 cells bearing plasmids expressing Asrij, Rab5-RFP, and STAT-FLAG were analyzed by coimmunolocalization of Rab5, STAT3 (FLAG), and Asrij. Graphs show colocalizing pixels. (B) Western blot of mESC lysate subjected to differential fractionation shows Asrij primarily in the membrane fraction. CF, cytoplasmic fraction; MF, membrane fraction; NF, nuclear fraction. (C) Western blot analysis of lysate from cells expressing Asrij alone or with FLAG-STAT3 as indicated, subjected to coimmunoprecipitation by FLAG antibody and probed with STAT3 and Asrij antibodies to assess interaction. Lanes 1 and 3: input control; lane 4: beads alone; lanes 2 and 5: FLAG immunoprecipitation, showing coimmunoprecipitation of Asrij only in the presence of STAT3. (D) Blocking endocytosis abolishes STAT3 phosphorylation. Western blot analysis of the relative pSTAT3 level in +/+ cells treated with the PI3K inhibitor LY290042. (E) In situ PLA for Asrij and STAT3 in ESCs. Interaction of STAT3 with Asrij or fragments in control cells (+/+) or those overexpressing ArjN or ArjC (as indicated) is seen as white complexes. The graph represents the PLA complex dots/cell. Nuclei in (A) and (E) were viewed with DAPI staining (blue). Boxed regions in (A) and (E) are shown as a magnified inset below the respective panel, with the blue channel off. Statistical significance is indicated by *p < 0.01, ***p < 0.0001. Error bars show SD of the mean. Scale bar, 12.5 μm.