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. 2024 Apr 8;15(1):34.
doi: 10.1186/s13293-024-00603-5.

Sex-based differences in growth-related IGF1 signaling in response to PAPP-A2 deficiency: comparative effects of rhGH, rhIGF1 and rhPAPP-A2 treatments

María Del Mar Fernández-Arjona #  1   2 , Juan Antonio Navarro #  1   3 , Antonio Jesús López-Gambero #  1   4 , Marialuisa de Ceglia  1   3 , Miguel Rodríguez  1   5 , Leticia Rubio  1   5 , Fernando Rodríguez de Fonseca  1   2 , Vicente Barrios  6   7   8 , Julie A Chowen  6   7   8   9 , Jesús Argente  10   11   12   13   14 , Patricia Rivera  15   16 , Juan Suárez  17   18
Affiliations

Sex-based differences in growth-related IGF1 signaling in response to PAPP-A2 deficiency: comparative effects of rhGH, rhIGF1 and rhPAPP-A2 treatments

María Del Mar Fernández-Arjona et al. Biol Sex Differ. .

Abstract

Background: Children with pregnancy-associated plasma protein-A2 (PAPP-A2) mutations resulting in low levels of bioactive insulin-like growth factor-1 (IGF1) and progressive postnatal growth retardation have improved growth velocity and height following recombinant human (rh)IGF1 treatment. The present study aimed to evaluate whether Pappa2 deficiency and pharmacological manipulation of GH/IGF1 system are associated with sex-specific differences in growth-related signaling pathways.

Methods: Plasma, hypothalamus, pituitary gland and liver of Pappa2ko/ko mice of both sexes, showing reduced skeletal growth, and liver of these mice treated with rhGH, rhIGF1 and rhPAPP-A2 from postnatal day (PND) 5 to PND35 were analyzed.

Results: Reduced body and femur length of Pappa2ko/ko mice was associated with increases in: (1) components of IGF1 ternary complexes (IGF1, IGFBP5/Igfbp5, Igfbp3, Igfals) in plasma, hypothalamus and/or liver; and (2) key signaling regulators (phosphorylated PI3K, AKT, mTOR, GSK3β, ERK1/2 and AMPKα) in hypothalamus, pituitary gland and/or liver, with Pappa2ko/ko females having a more prominent effect. Compared to rhGH and rhIGF1, rhPAPP-A2 specifically induced: (1) increased body and femur length, and reduced plasma total IGF1 and IGFBP5 concentrations in Pappa2ko/ko females; and (2) increased Igf1 and Igf1r levels and decreased Ghr, Igfbp3 and Igfals levels in the liver of Pappa2ko/ko females. These changes were accompanied by lower phospho-STAT5, phospho-AKT and phospho-ERK2 levels and higher phospho-AMPK levels in the liver of Pappa2ko/ko females.

Conclusions: Sex-specific differences in IGF1 system and signaling pathways are associated with Pappa2 deficiency, pointing to rhPAPP-A2 as a promising drug to alleviate postnatal growth retardation underlying low IGF1 bioavailability in a female-specific manner.

Keywords: AMPK; GH; Growth; Hypothalamus; IGF1; Liver; PI3K; Pappalysin-2; Pituitary gland; Sexual dimorphism; mTOR.

Plain language summary

Understanding the physiological role of pregnancy-associated plasma protein-A2 (PAPP-A2), a proteinase involved in the insulin-like growth factor-1 (IGF1) availability to regulate growth, could provide insight into new treatments for patients with short stature and skeletal abnormalities. Although progressive postnatal growth retardation in patients with PAPP-A2 mutations can differ between males and females, we do not know the underlying differences in IGF1 system and signaling, and their response to treatment that contribute to growth improvement. The present study examines whether Pappa2 deficiency and pharmacological administration of rhGH, rhIGF1 and rhPAPP-A2 are associated with sex-specific differences in IGF1 ternary complexes and IGF1 signaling pathways. Reduced body and femur length of Pappa2-deficient mice was associated with sex- and tissue-specific alteration of IGF ternary/binary complexes and IGF1 signaling pathways. rhPAPP-A2 treatment induced female-specific increase in body and femur length and reduction in IGF ternary/binary complexes through STAT5-AKT-ERK2-AMPK signaling pathways in liver. The involvement of PAPP-A2 in sex-based growth physiology supports the use of promising drugs to alleviate postnatal growth retardation underlying low IGF1 bioavailability in a female-specific manner.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Fig. 1
Fig. 1
Auxological parameters of body and bone (femur and tibia) length in Pappa2ko/ko and Pappa2wt/wt mice (males and females) at weaning (A), puberty (B) and adulthood (C-G). Data are represented as mean ± S.E.M (N = 8–10 per experimental group). Two-way ANOVA and Tukey-corrected tests: ***p < 0.001 versus respective wt/wt males and females; #/##/###p < 0.05/0.01/0.001 versus respective wt/wt and ko/ko males
Fig. 2
Fig. 2
Hormone concentrations in the plasma and pituitary gland of Pappa2ko/ko and Pappa2wt/wt mice (males and females) in adulthood. Concentrations of total IGF1 (A), IGFBP5 (B) and GH in plasma (C), and GH in pituitary gland (D) are represented as mean ± S.E.M (n = 8–10 per experimental group). Two-way ANOVA and Tukey-corrected tests: */***p < 0.05/0.001 versus respective wt/wt males and females; ###p < 0.001 versus respective wt/wt and ko/ko males
Fig. 3
Fig. 3
IGF1 system gene expression in the hypothalamus, the pituitary gland and the liver of Pappa2ko/ko and Pappa2wt/wt mice (males and females) in adulthood. Hypothalamus: relative Pappa2 (A), Ghrh (B), Ghih-Sst (C), Igf1 (D), Igf1r (E), Igfbp3 (F), Igfbp5 (G), Igfals (H) and Stc1 (I) mRNA levels. Pituitary gland: relative Pappa2 (J), Gh (K), Ghrhr (L), Igf1 (M), Igf1r (N), Igfbp3 (O), Igfbp5 (P), Igfals (Q) and Stc1 (R) mRNA levels. Liver: relative Ghr (S), Igf1 (T), Igf1r (U), Igf2 (V), Igfbp3 (W), Igfbp5 (X), Igfals (Y) and Stc1 (Z) mRNA levels. Data are represented as mean ± S.E.M (n = 8–10 per experimental group). See Table S1 for additional information. Two-way ANOVA and Tukey-corrected tests: */***p < 0.05/0.001 versus respective wt/wt males and females; ##/###p < 0.01/0.001 versus respective wt/wt and ko/ko males
Fig. 4
Fig. 4
Key sensors of IGF1 signaling pathways in the hypothalamus, the pituitary gland and the liver of Pappa2ko/ko and Pappa2wt/wt mice (males and females) in adulthood. Hypothalamus: phosphorylated forms of PI3K (A), AKT (B), mTOR (C), GSK3β (D), ERK1 (E), ERK2 (F), AMPKα (G) and GS (H). Pituitary gland: phosphorylated forms of GSK3β (J), AMPKα (K), ERK1 (L) and ERK2 (M). Liver: phosphorylated forms of PI3K (O), AKT (P), mTOR (Q), GSK3β (R), ERK1 (S), ERK2 (T), AMPKα (U) and GS (V). Data are represented as mean ± S.E.M (n = 6 per experimental group). Representative immunoblots (I, N and W) are also shown. See Table S2 and Figures S1-S3 for additional information. Two-way ANOVA and Tukey-corrected tests: */**/***p < 0.05/0.01/0.001 versus respective wt/wt males and females; #/##/###p < 0.05/0.01/0.001 versus respective wt/wt and ko/ko males
Fig. 5
Fig. 5
Experimental design (A) and auxological parameters of body (B), femur (C) and tibia (D) length in Pappa2ko/ko and Pappa2wt/wt mice (males and females) treated with rhGH, rhIGF1 and rhPAPP-A2 for 30 days, from PND5 to PND35. Data are represented as mean ± S.E.M (N = 8–10 per experimental group). Two-way ANOVA and Tukey-corrected tests: */**/***p < 0.05/0.01/0.001 versus respective saline-treated males and females; #/##/###p < 0.05/0.01/0.001 versus respective wt/wt males and females
Fig. 6
Fig. 6
Experimental design (A) and schematic diagram (B) of the analysis of circulating hormone concentrations, and liver IGF1 system gene expression in Pappa2ko/ko and Pappa2wt/wt mice (males and females) treated with rhGH, rhIGF1 and rhPAPP-A2 for 30 days, from PND5 to PND35. Concentrations of total IGF1 (C) and IGFBP5 (D) in plasma, and relative Igf1 (E), Ghr (F), Igf1r (G), Igfbp3 (H), Igfbp5 (I), Igfals (J), Stc1 (K) and Stc1 (L) mRNA levels are represented as mean ± S.E.M (n = 8–10 per experimental group). See Table S1 for additional information. Two-way ANOVA and Tukey-corrected tests: */**/***p < 0.05/0.01/0.001 versus respective saline-treated males and females; #/##/###p < 0.05/0.01/0.001 versus respective wt/wt males and females
Fig. 7
Fig. 7
Experimental design (A) and schematic diagram (B) of the analysis of key regulators of IGF1 signaling pathways in the liver of Pappa2ko/ko and Pappa2wt/wt mice (males and females) treated with rhGH, rhIGF1 and rhPAPP-A2 for 30 days, from PND5 to PND35. Phosphorylated forms of IRS1 (C), PI3K (D), AKT (E), mTOR (F), GSK3β (G), ERK2 (H) and AMPKα (I) are represented as mean ± S.E.M (n = 6 per experimental group). Representative immunoblots (J) are also shown. See Table S2 and Figures S4 and S6 for additional information. Two-way ANOVA and Tukey-corrected tests: */**/***p < 0.05/0.01/0.001 versus respective saline-treated males and females; #/##p < 0.05/0.01 versus respective wt/wt males and females
Fig. 8
Fig. 8
Experimental design (A) and schematic diagram (B) of the analysis of key regulators of GH signaling pathways in the liver of Pappa2ko/ko and Pappa2wt/wt mice (males and females) treated with rhGH, rhIGF1 and rhPAPP-A2 for 30 days, from PND5 to PND35. Phosphorylated forms of JAK2 (C), STAT3 (D) and STAT5 (E) are represented as mean ± S.E.M (n = 6 per experimental group). Representative immunoblots (F) are also shown. See Table S2 and Figures S4 and S7 for additional information. Two-way ANOVA and Tukey-corrected tests: */**p < 0.05/0.01 versus respective saline-treated males and females; #/##p < 0.05/0.01 versus respective wt/wt males and females
Fig. 9
Fig. 9
(A) Schematic diagrams summarizing the alterations in the hypothalamus-pituitary-liver axis that result in decreased body and femur length in Pappa2ko/ko mice (males and females) in adulthood. Increased and decreased levels are represented by a blue arrow for males and a red arrow for females. The effect of genotype is indicated by an asterisk (*). Reduced body and femur length of Pappa2ko/ko mice and female-specific increased expressions of components of IGF1 binary/ternary complexes (IGF1 and IGFBP5 in plasma, Igfbp5 in hypothalamus, and Igfbp3 and Igfals in liver) could be consistent with reduced IGF1 availability and signaling in _target tissues. Sex-specific modulation of key sensors of IGF1 signaling pathways (phosphorylation of AKT, mTOR, GSK3β and ERK1/2 in the female hypothalamus, phosphorylation of GSK3β and ERK1/2 in the male pituitary gland, and phosphorylation of PI3K and AMPKα in the female liver) may be relevant to explain growth impairment through a dysregulated IGF1 system due to PAPP-A2 deficiency. (B) Schematic diagrams summarizing changes in IGF1 ternary complex components and IGF1 signaling pathways that result in increased body and/or femur length after the pharmacological administration of rhGH, rhIGF1 and rhPAPP-A2 for 30 days (PND5-35) in Pappa2ko/ko mice (males and females) at puberty onset. Increased and decreased levels are represented by a blue arrow for males and a red arrow for females. The effect of treatment is indicated by an asterisk (*). Female-specific increases in body and femur length and female-specific decreases in expressions of components of IGF1 ternary complexes (IGF1 and IGFBP5 in plasma, and Igfbp3 and Igfals in liver) could be consistent with increased IGF1 availability and signaling in _target tissues. Female-specific modulation of key sensors of liver IGF1 signaling pathways (reduced phosphorylation of AKT and ERK2, and increased phosphorylation of AMPKα) may be relevant to explain growth improvement through a regulated IGF1 system by rhPAPP-A2 treatment
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