The IRE1α-XBP1 pathway mediates chemotherapy-induced hepatic GDF15 upregulation. (A and B) Western blot analysis of the indicated proteins (left) and RT-qPCR analysis of GDF15 mRNA abundance (right) in cell lysates of Huh7 cells transfected with control vector, or vector expressing IRE1α (OE-IRE1α) (A) or XBP1s (OE-XBP1s) (B). Tubulin was used as a loading control. (C)GDF15 mRNA levels in Huh7 cells transfected with control siRNA (si-NC) or ERN1 knockdown siRNA (si-ERN1) prior to treatment with DOX (0.01 μg/ml) or Cis (10 μM) for 48 h. n = 3 per group. (D) Schematic of the luciferase constructs of Gdf15 promoter with the ERSE-like sequence indicated. Luciferase reporter assays were performed by co-transfection of HEK293T cells with the control vector or pCMV-XBP1s plasmid together with Luc constructs under the control of the mouse Gdf15 promoter (WT) or those with mutant ERSE sequences (Mut1, Mut2, Mut3). n = 3 per group. (E) ChIP-qPCR analysis of XBP1s binding site on Gdf15 promoter was performed by overexpression of XBP1s in primary hepatocytes (n = 4). ChIP-qPCR of the segments containing XBP1s-binding site (−713 to −515) or no XBP1s-binding site (−1,459 to −1,327) within the Gdf15 promoter. (F)flox/flox (littermates) and LKO mice were engineered to express the empty control construct or the Gdf15-WT Luc reporter in livers. Luciferase activities were monitored in vivo by the imaging system under the indicated treatments. Mice were i.p. administered with DOX (5 mg per kg body weight) or Cis (5 mg per kg body weight) 24 h prior to luciferase assays. (G and H)Xbp1 mRNA splicing (G) and mRNA levels of Gdf15 (H) in livers from flox/flox and LKO mice following DOX treatment (5 mg per kg body weight, i.p.) for 1 day. flox/flox+Veh., n = 4; LKO+Veh., n = 5; flox/flox+DOX, n = 5; LKO+DOX, n = 7. (I) Circulating GDF15 protein levels in flox/flox (n = 7) and LKO (n = 7) mice at the indicated time points following DOX treatment (5 mg per kg body weight, i.p.). (J) Serum GDF15 protein levels in non-tumor-bearing (Non T.B.) flox/flox (n = 12) and LKO (n = 12) mice, and in tumor-bearing (T.B.) flox/flox and LKO mice treated with Vehicle (Veh.; n = 12 for flox/flox, n = 10 for LKO) or DOX (5 mg per kg body weight, i.p., n = 12 for each group) for 1 day. (K) Representative images of immunofluorescence staining against c-Fos and GFRAL at the AP and NTS of the murine brainstem (left). GFRAL+ c-Fos+ cells (indicated by arrowheads, left) in AP or NTS per high power field (HPF) were quantified (right). The frozen brainstem slides were from flox/flox and LKO mice at 1 day after DOX treatment (5 mg per kg body weight, i.p.). Scale bar, 100 μm. (L and M)Xbp1 mRNA splicing (L) and mRNA levels of Gdf15 (M) in liver samples from flox/flox and LKO mice following Cis treatment (5 mg per kg body weight, i.p.) for 1 day. flox/flox+Veh., n = 4; LKO+Veh., n = 5; flox/flox+Cis, n = 5; LKO+Cis, n = 5. (N) Circulating GDF15 protein levels in flox/flox (n = 10) and LKO (n = 10) mice at the indicated time points following Cis treatment (5 mg per kg body weight, i.p.). (O) Representative images of immunofluorescence staining against c-Fos and GFRAL at the area AP and NTS of the murine brainstem (left). GFRAL+ c-Fos+ cells (indicated by arrowheads, left) per HPF were quantified (right). The frozen brainstem slides were from flox/flox and LKO mice at 1 day after Cis treatment (5 mg per kg body weight, i.p.). Scale bar, 100 μm. Data are representative of three independent experiments (A–G) or two independent experiments (H–P) and presented as mean ± SEM. *P < 0.05, **P < 0.01, or ***P < 0.001 by unpaired two-tailed Student’s t test (A, B, I, K, and O) or two-way ANOVA (C–H, J, L–N). Source data are available for this figure: SourceData F4.
Figure 4.

The IRE1α-XBP1 pathway mediates chemotherapy-induced hepatic GDF15 upregulation. (A and B) Western blot analysis of the indicated proteins (left) and RT-qPCR analysis of GDF15 mRNA abundance (right) in cell lysates of Huh7 cells transfected with control vector, or vector expressing IRE1α (OE-IRE1α) (A) or XBP1s (OE-XBP1s) (B). Tubulin was used as a loading control. (C)GDF15 mRNA levels in Huh7 cells transfected with control siRNA (si-NC) or ERN1 knockdown siRNA (si-ERN1) prior to treatment with DOX (0.01 μg/ml) or Cis (10 μM) for 48 h. n = 3 per group. (D) Schematic of the luciferase constructs of Gdf15 promoter with the ERSE-like sequence indicated. Luciferase reporter assays were performed by co-transfection of HEK293T cells with the control vector or pCMV-XBP1s plasmid together with Luc constructs under the control of the mouse Gdf15 promoter (WT) or those with mutant ERSE sequences (Mut1, Mut2, Mut3). n = 3 per group. (E) ChIP-qPCR analysis of XBP1s binding site on Gdf15 promoter was performed by overexpression of XBP1s in primary hepatocytes (n = 4). ChIP-qPCR of the segments containing XBP1s-binding site (−713 to −515) or no XBP1s-binding site (−1,459 to −1,327) within the Gdf15 promoter. (F)flox/flox (littermates) and LKO mice were engineered to express the empty control construct or the Gdf15-WT Luc reporter in livers. Luciferase activities were monitored in vivo by the imaging system under the indicated treatments. Mice were i.p. administered with DOX (5 mg per kg body weight) or Cis (5 mg per kg body weight) 24 h prior to luciferase assays. (G and H)Xbp1 mRNA splicing (G) and mRNA levels of Gdf15 (H) in livers from flox/flox and LKO mice following DOX treatment (5 mg per kg body weight, i.p.) for 1 day. flox/flox+Veh., n = 4; LKO+Veh., n = 5; flox/flox+DOX, n = 5; LKO+DOX, n = 7. (I) Circulating GDF15 protein levels in flox/flox (n = 7) and LKO (n = 7) mice at the indicated time points following DOX treatment (5 mg per kg body weight, i.p.). (J) Serum GDF15 protein levels in non-tumor-bearing (Non T.B.) flox/flox (n = 12) and LKO (n = 12) mice, and in tumor-bearing (T.B.) flox/flox and LKO mice treated with Vehicle (Veh.; n = 12 for flox/flox, n = 10 for LKO) or DOX (5 mg per kg body weight, i.p., n = 12 for each group) for 1 day. (K) Representative images of immunofluorescence staining against c-Fos and GFRAL at the AP and NTS of the murine brainstem (left). GFRAL+ c-Fos+ cells (indicated by arrowheads, left) in AP or NTS per high power field (HPF) were quantified (right). The frozen brainstem slides were from flox/flox and LKO mice at 1 day after DOX treatment (5 mg per kg body weight, i.p.). Scale bar, 100 μm. (L and M)Xbp1 mRNA splicing (L) and mRNA levels of Gdf15 (M) in liver samples from flox/flox and LKO mice following Cis treatment (5 mg per kg body weight, i.p.) for 1 day. flox/flox+Veh., n = 4; LKO+Veh., n = 5; flox/flox+Cis, n = 5; LKO+Cis, n = 5. (N) Circulating GDF15 protein levels in flox/flox (n = 10) and LKO (n = 10) mice at the indicated time points following Cis treatment (5 mg per kg body weight, i.p.). (O) Representative images of immunofluorescence staining against c-Fos and GFRAL at the area AP and NTS of the murine brainstem (left). GFRAL+ c-Fos+ cells (indicated by arrowheads, left) per HPF were quantified (right). The frozen brainstem slides were from flox/flox and LKO mice at 1 day after Cis treatment (5 mg per kg body weight, i.p.). Scale bar, 100 μm. Data are representative of three independent experiments (A–G) or two independent experiments (H–P) and presented as mean ± SEM. *P < 0.05, **P < 0.01, or ***P < 0.001 by unpaired two-tailed Student’s t test (A, B, I, K, and O) or two-way ANOVA (C–H, J, L–N). Source data are available for this figure: SourceData F4.

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