Kumar S et al. (2024), Xbra modulates the activity of linker region ph...

XB-ART-60730

Sci Rep 2024 Apr 18;141:8922. doi: 10.1038/s41598-024-59299-7.

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Xbra modulates the activity of linker region phosphorylated Smad1 during Xenopus development.

Kumar S , Umair Z , Kumar V , Goutam RS , Park S , Lee U , Kim J .

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The Bmp/Smad1 pathway plays a crucial role in developmental processes and tissue homeostasis. Mitogen-activated protein kinase (Mapk)/Erk mediated phosphorylation of Smad1 in the linker region leads to Smad1 degradation, cytoplasmic retention and inhibition of Bmp/Smad1 signaling. While Fgf/Erk pathway has been documented to inhibit Bmp/Smad1 signaling, several studies also suggests the cooperative interaction between these two pathways in different context. However, the precise role and molecular pathway of this collaborative interaction remain obscure. Here, we identified Xbra induced by Fgf/Erk signaling as a factor in a protective mechanism for Smad1. Xbra physically interacted with the linker region phosphorylated Smad1 to make Xbra/Smad1/Smad4 trimeric complex, leading to Smad1 nuclear localization and protecting it from ubiquitin-mediated proteasomal degradation. This interaction of Xbra/Smad1/Smad4 led to sustained nuclear localization of Smad1 and the upregulation of lateral mesoderm genes, while concurrently suppression of neural and blood forming genes. Taken together, the results suggests Xbra-dependent cooperative interplays between Fgf/Erk and Bmp/Smad1 signaling during lateral mesoderm specification in Xenopus embryos.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 fgf4 myc smad1 smad4 tbxt
GO keywords: mesodermal cell fate commitment

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	Figure 1. Influence of eFGF on smad1 nuclear localization and gene expression in BMP4-treated animal cap explants. (A) Subcellular localization of smad1 in Xenopus animal cap explant in the presence of control, BMP4 and BMP4 with eFGF injected. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay with primary antibody against endogenous smad1 and anti-rabbit Alexa-fluor 594 secondary antibody (Invitrogen) was used. Blue: DAPI, Red: endogenous smad. Scale bar ×20. This experiment was repeated two times with consistent results. Right: Statistic data of smad1 nuclear translocation ratio. (B) Subcellular localization of flag-smad1 in Xenopus animal cap explant in the presence of control flag-smad1, flag-smad1 with BMP4 and flag-smad1 with BMP4 and eFGF injected. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay with primary antibody against endogenous smad1 and anti-mouse Alexa-fluor 488 secondary antibody (Invitrogen) was used. Blue: DAPI, Green: flag tag. Scale bar ×20. This experiment was repeated two times with consistent results. Right: Statistic data of Flag-smad1 nuclear translocation ratio. (C) Animal cap from the embryos injected with Flag-Smad1, BMP4 and eFGF were used to fractionate cytoplasmic and nuclear lysate to perform WB analysis using anti-flag antibody. (D–Q) qRT-PCR Analysis of the gene expression levels of early and late markers in animal cap explants from embryos injected with eFGF and BMP4 together or separately. qRT-PCR values were determined from the ΔΔCt for the target genes relative to odc. Significantly different results for treatments used one-way ANOVA with Graph Pad Prism; P < 0.05.
	Figure 2. Synergistic enhancement of Smad1/Xbra complex formation through eFgf and Bmp4 stimulation. (A) Animal cap from embryos injected with myc-xbra and eFGF either treated with U0126 or control DMSO and harvested for co-immunoprecipitation analysis using endogenous smad1 and anti-myc antibodies. (B) Animal cap from embryos injected with flag-smad1 and myc-xbra either treated with U0126 or control DMSO and harvested for co-immunoprecipitation analysis using anti-flag and anti-myc antibodies. α-tubulin served as loading control. Right: Flag-smad1 level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (C) Immunoblotting analysis of total lysates from animal cap explant from embryo injected with flag-smad1 together with or without eFGF and BMP4 lysates were analyzed by immunoblotting using p-smad1linker206, p-smad1linker214, p-smad1/5/9c-terminal and p-Erk antibody. Pan Erk and α-tubulin served as a loading control. (D) Smad1 phospho-mutant forms, constitutively active FGF- and BMPR-mediated phosphorylated form of flag-smad1(4SD3SD), and constitutively inactive FGF- and active BMPR-mediated phosphorylated form of flag-smad1(4SA3SD) along with wild type. (E) Embryos injected with flag-smad1 (WT), flag-smad1(4SD3SD) or flag-smad1(4SA3SD) with myc-xbra and harvested for co-immunoprecipitation analysis using anti-flag and anti-myc antibodies. Right: Myc-Xbra level were quatified by photo shop software. This experiment was repeted two times with consistemce results.
	Figure 3. The nuclear localization and induction of lateral mesoderm markers by the phospho-mimetic Smad1 mutant, mediated by Fgf and Bmp, require Xbra. (A) Subcellular localization of flag-smad1(4SD3SD) in Xenopus animal cap explant from embryos injected with flag-smad1(4SD3SD) and myc-xbra together and separately. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay using primary antibody against flag-tag and myc-tag. Anti-mouse Alexa-fluor 488 and anti-rabbit Alexa fluor 594 secondary antibody (Invitrogen) was used. Blue: DAPI, Green: flag tag, Red: myc-tag. Scale bar ×20. This experiment was repeated two times with consistent results. Right: Statistic data of smad1 nuclear translocation ratio. (B) Subcellular localization of flag-smad1 in Xenopus animal cap explant from embryos injected with flag-smad1, DNXbra, myc-xbra along with BMP4 and eFGF with indicated combinations. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay using primary antibody against flag-tag and myc-tag. Anti-mouse Alexa-fluor 488 and anti-rabbit Alexa fluor 594 secondary antibody (Invitrogen) was used. Blue: DAPI, Green: flag tag, Red: myc-tag. Scale bar ×20. (C–L) qRT-PCR Analysis of the gene expression levels of early and late markers in animal cap explants from embryos injected with smad1(WT) and smad1(4SD3SD) with or without xbra. qRT-PCR values were determined from the ΔΔCt for the target genes relative to odc. Significantly different results for treatments used one-way ANOVA with Graph Pad Prism; P < 0.05.
	Figure 4. Xbra inhibits the polyubiquitination and degradation of endogenous Smad1 as well as a phospho-mimetic Smad1 Mutant. (A) Embryos injected with flag-ubiquitin with or without myc-xbra harvested at stage 10.5 for co-immunoprecipitation analysis using anti-smad1 and anti-flag antibodies. α-tubulin served as loading control. Right: Flag-Ubiquitin level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (B) Embryos injected with HA-smad1(WT) and Flag-ubiquitin with or without myc-xbra harvested at stage 10.5 for co-immunoprecipitation analysis using anti-ha and anti-flag antibodies. α-tubulin served as loading control. Right: Flag-Ubiquitin level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (C) Embryos injected with Flag-smad1(4SD3SD) with or without myc-xbra harvested at stage 10.5 for co-immunoprecipitation analysis using anti-flag and anti-ubiquitin antibodies. Right: Endogenous Ubiquitin level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (D) Embryos injected with flag-Smad1, DNXbra and Myc-Xbra alongwith BMP4 and eFGF embryos were harvested at the stage of 10.5 for co-immunoprecipitation analysis using anti-flag and ubiquitin antibody. (E) Embryos injected with myc-xbra and ubiquitin alone or in combination were treated with 200 μM/ml of cycloheximide at stage 10.5 and harvested at 0 h. 3 h. and 6 h. for WB analysis using anti smad1 antibody. α-tubulin served as loading control. Right: Endogenous smad1 level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (F) Embryos injected with flag-smad1(4SD3SD) with or without myc-xbra were treated with 200 μM/ml of cycloheximide at stage 10.5 and harvested at 0 h. 3 h. and 6 h. for WB analysis using anti-flag antibody. α-tubulin served as loading control. Right: Flag-smad1 level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (G) Embryos injected with flag-smad1(4SD3SD), myc-xbra and myc-xbra alone or in combination were treated with 200 μM/ml of cycloheximide at stage 10.5 and harvested at 0 h. 2 h. and 4 h. for WB analysis using anti-flag antibody. α-tubulin served as loading control. (H) Embryos injected with flag-smad1(4SD3SD) and with or without xbra were treated with 200 μM/ml of cycloheximide and Licl at stage of 10.5 and harvested at 0 h. 2 h. and 4 h. for WB analysis using anti-flag antibody. α-tubulin served as loading control.
	Figure. 5 Smad4 plays a crucial role in the formation of the Smad1/Xbra complex, and it is essential for facilitating the nuclear localization and transcription activity of the phospho-mimetic Smad1 mutant mediated by Xbra. (A) Embryos injected with smad4MO harvested at stage 10.5 and subjected for WB analysis using anti-smad4 antibody. (B) Embryos injected with myc-xbra and smad4 with and without smad4MO harvested at stage 10.5 for co-immunoprecipitation analysis using anti-smad1 and anti-myc antibodies. α-tubulin served as loading control. Right: Myc-Xbra level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (C) Embryos injected with Flag-smad1(4SD3SD), myc-xbra and smad4 with and without smad4MO harvested at stage 10.5 for co-immunoprecipitation analysis using anti-Flag and anti-myc antibodies. Right down: Myc-Xbra level were quatified by photo shop software. This experiment was repeted two times with consistemce results. (D) Subcellular localization of flag-smad1(4SD3SD) in Xenopus animal cap explant from embryos injected with flag-smad1(4SD3SD), myc-xbra and smad4 with or without smad4MO. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay using primary antibody against flag-tag and myc-tag. Anti-mouse Alexa-fluor 488 and anti-rabbit Alexa fluor 594 secondary antibody (Invitrogen) was used. Blue: DAPI, Green: flag tag, Red: myc-tag. Scale bar ×20. This experiment was repeated two times with consistent results. Right: Statistic data of smad1 nuclear translocation ratio. (E) Subcellular localization of flag-smad1(4SD3SD) in Xenopus animal cap explant from embryos injected with flag-smad1(4SD3SD), smad4, smad4MO and Myc-Xbra in the shown combination. Animal cap dissected at 9.5 stage and grown until stage 10.5, fixed and then subjected to immunofluorescence assay using primary antibody against flag-tag and myc-tag. Anti-mouse Alexa-fluor 488 and anti-rabbit Alexa fluor 594 secondary antibody (Invitrogen) was used. Blue: DAPI, Green: flag tag, Red: myc-tag. Scale bar ×20. (F–I) qRT-PCR Analysis of the gene expression levels of early markers in animal cap explants from embryos injected with smad1(4SD3SD), xbra and smad4 with or without smad4MO. qRT-PCR values were determined from the ΔΔCt for the target genes relative to odc. Significantly different results for treatments used one-way ANOVA with Graph Pad Prism; P < 0.05. (G,H) qRT-PCR Analysis of the gene expression levels of late markers in animal cap explants from embryos injected with smad1(4SD3SD), xbra and smad4 with or without smad4MO. qRT-PCR values were determined from the ΔΔCt for the target genes relative to odc. Significantly different results for treatments used one-way ANOVA with Graph Pad Prism; P < 0.05.
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References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase