Sex-based differences in IGF1 signaling pathways in response to PAPP-A2 deficiency

doi:10.21203/rs.3.rs-2489074/v1

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Sex-based differences in IGF1 signaling pathways in response to PAPP-A2 deficiency

https://doi.org/10.21203/rs.3.rs-2489074/v1

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Background.

Patients with pregnancy-associated plasma protein-A2 (PAPP-A2) mutations have progressive postnatal growth retardation and high circulating levels of IGF1 bound in ternary complexes. The present study aims to assess whether Pappa2 deficiency is associated with sex-specific differences in the main components of IGF1 ternary complexes and IGF1 signaling pathways in response to low IGF1 bioavailability.

Methods.

Plasma, hypothalamus, pituitary gland and liver were analyzed in constitutive Pappa2^ko/ko mice of both sexes that have reduced skeletal growth and impaired bone composition.

Results.

The reduction in body and femur length of Pappa2^ko/ko mice was associated with increases in total IGF1 and IGFBP5 concentrations in plasma of females, Igfbp5 mRNA levels in the hypothalamus of males, and Igf1, Igfbp3 and Igfals mRNA levels in the liver of females, suggesting sex- and tissue-specific effects of Pappa2 deficiency on IGF ternary/binary complexes. Pappa2 deficiency was also accompanied by increased pituitary GH concentrations in both sexes. Sex-specific dysregulation of IGF1 signaling pathways was found in Pappa2^ko/ko mice with higher phosphorylated forms of AKT, mTOR, GSK3β and ERK1/2 in the female hypothalamus, GSK3β in the male pituitary gland, and PI3K and AMPKα in the female liver, suggesting sex-based alterations in regulators of cell proliferation/growth and protein/glucose metabolism.

Conclusions.

These data suggest that sex-specific differences in IGF ternary complexes and IGF1 signaling pathways are associated with Pappa2 deficiency, pointing to molecular mechanisms that may participate in the physiopathology of postnatal growth retardation in a sex-dependent manner.

Growth

sexual dimorphism

pappalysin-2

IGF1

PI3K

mTOR

AMPK

hypothalamus

pituitary gland

liver

Homozygous mutations in pregnancy-associated plasma protein-A2 (PAPP-A2 or pappalysin-2) produce a syndrome characterized by progressive postnatal growth retardation, loss or a reduction of PAPP-A2 function, high circulating levels of insulin-like growth factor (IGF)-1 bound in ternary/binary complexes with specific IGF binding proteins (IGFBP3 or IGFBP5) and acid-labile subunit (IGFALS), and decreased concentrations of free IGF1 [1, 2]. Patients with PAPP-A2 mutations have mild to moderate short stature and skeletal abnormalities including thin long bones and decreased bone mineral density [3, 4] and when treated with recombinant human IGF1 (rhIGF1) both growth [5] and bone mineral density [6] improve.

PAPP-A2 is a metalloproteinase that exerts specific proteolytic activity on IGFBP3 and IGFBP5 and regulates IGF1 dissociation from ternary/binary complexes, and thus IGF1 bioavailability [7, 8]. Inhibitors of PAPP-A2 activity, staniocalcin-1 (STC1) and staniocalcin-2 (STC2), block IGF release from IGFBP3 and IGFBP5, bringing another level of complexity to the control of IGF1 bioavailability [9, 10, 11]. In fact, serum concentrations of PAPP-A2 and the free/total IGF1 ratio, but not stanniocalcins, decrease progressively in an age-specific manner [12]. Synthesis and secretion of IGF1, among other IGF-related components such as IGF2, IGFBP3, IGFBP5 and IGFALS, are stimulated by the actions of growth hormone (GH) in the liver [3, 4]. IGF1 primarily circulates in ternary complexes (75–80% of total IGF1) with the high affinity IGFBP3 or IGFBP5 and IGFALS, which increases its half-life and modulates its concentrations and bioavailability [13,14,15], with only approximately 1% being free or unbound [16]. IGF1 is liberated from these binding proteins by PAPP-A2 in close proximity to target tissues that contain the IGF1 receptor (IGF1R).

PAPP-A2 actions increase the levels of free IGF1 and the subsequent availability of this growth factor to stimulate its receptor both centrally and peripherally that, in conjunction with an adequate nutritional supply, is predominately responsible for activating and/or inhibiting key signaling pathways and transcriptional factors that modulate growth. IGF1R intracellular signaling pathways include phosphoinositide 3-kinase (PI3K)/ protein kinase B (PKB, also known AKT), mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK3), among others [17,18,19].

Preclinical studies using knock-out (ko/ko) and knock-in mice with targeted disruption of PAPP-A2 [20,21,22,23] have emerged as good experimental models to recapitulate previously described physiological growth derangements related to short stature in patients with PAPP-A2 deficiency [4]. However, more information is needed to identify specific signaling mechanisms affected by low IGF1 availability and to elucidate whether impairment of postnatal longitudinal growth caused by increased IGF1 ternary/binary complexes and reduced IGF1 availability are different between the sexes. To this aim, plasma, hypothalamus, pituitary gland and liver of constitutive Pappa2^ko/ko mice of both sexes with reduced skeletal growth and impaired bone composition, as previously described by our group and others [23,24], were used to assess sex-specific differences in the expression of the main components of IGF1 ternary complexes and main mediators of IGF1 intracellular signaling pathways (PI3K/AKT, mTOR, GSK3β, ERK1/2 and AMPK).

Ethics statement

All procedures were conducted in strict adherence to ARRIVE guidelines and the principles of laboratory animal care (National Research Council, Neuroscience CoGftUoAi, Research B, 2003) following the European Community Council Directive (86/609/EEC) and were approved by the Ethical Committee of the University of Málaga (Exp. 46-2019-A). Special care was taken to minimize suffering and the number of animals required to perform the procedures.

Animals

Male and female mice with constitutive deletion of the Pappa2 gene (Pappa2^ko/ko) and littermate controls (Pappa2^wt/wt) were generated as previously described [21]. Pappa2 lines (C57BL/6 genetic background) were maintained by crossing heterozygous transgenic mice. The mice were housed in the Animal Center for Experimentation at the University of Malaga with free access to standard rodent chow (3.02 Kcal/g) and tap water under standardized conditions: 22 ± 1 ºC of room temperature, relative humidity of 40 ± 5%, and a 12-h reverse light/dark cycle (lights were turned off at 8:00 am) with dawn/sunset effect. Mice were genotyped using ear clip tissue as previously described [8]. Body length (including tail) was monitored periodically from weaning (approximately PND23) to adulthood (8 months of age).

Sample collection

Mice with 8 months of age were sacrificed by decapitation after administration of Equitesin® (3 ml/kg i.p.; chloral hydrate 2.1 g, sodium pentobarbital 0.46 g, MgSO₄ 1.06 g, propylene glycol 21.4 ml, ethanol (90%) 5.7 ml, H₂O 3 ml). Blood samples were collected in tubes containing 6% EDTA and centrifuged (1500 g for 10 min at 4ºC). The plasma was kept at -80 ºC until hormone analysis. Bones (femurs and tibias) were weighed, and their length was measured with a caliper. Brain, pituitary gland and samples from the left lateral lobe of the liver were extracted, flash-frozen in liquid nitrogen and stored at -80 ºC until used for mRNA and protein analysis. The hypothalamus, containing the median eminence, arcuate nucleus and ventromedial nucleus, were precisely dissected according to the mouse brain atlas of Paxinos and Franklin [25].

Hormone analyses

Plasma levels of IGF1 and IGFBP5 were determined by using commercial enzyme-linked immunosorbent assay (ELISA) kits: Mouse/rat IGF1 DuoSet® ELISA (cat. no. DY791, R&D Systems, Abingdon, UK) and mouse IGFBP5 DuoSet® ELISA (cat. no. DY578, R&D Systems). Plasma and pituitary levels of growth hormone (GH) were determined by using the Mouse Pituitary Magnetic Bead Panel 96-well Plate Assay (cat. no. MPTMAG-49K, Millipore® Map, Merck). Plates were run on a Bio-Plex MAGPIX™ Multiplex Reader with Bio-Plex manager™ MP Software (Luminex, Austin, TX, USA). Hormone concentrations were expressed in ng/mL or pg/mL, and detection limits were 31.3 (IGF1), 125 (IGFBP5) and 3.9 (GH) pg/mL.

RNA isolation and RT-qPCR analysis

Real-time PCR (TaqMan, ThermoFisher Scientific, Waltham, MA, USA) was performed as previously described [26] by using specific sets of primer probes from TaqMan® Gene Expression Assays (Table 1). Hypothalamus, pituitary and liver samples were homogenized on ice and total RNA was extracted using the Trizol® method according to the manufacturer’s instructions (ThermoFisher Scientific). RNA samples were isolated with RNAeasy minielute cleanup kits, including digestion with DNase I column (Qiagen), and quantified using a spectrophotometer to ensure A260/280 ratios of 1.8-2.0. After the reverse transcript reaction from 1 µg of RNA, a quantitative real-time reverse transcription polymerase chain reaction (qPCR) was performed in a CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the FAM dye-labeled format for the TaqMan® Gene Expression Assays (ThermoFisher Scientific). Melting curve analysis was performed to ensure that only a single product was amplified. After analysis of several reference genes, the values obtained from the samples were normalized to Actb levels, which did not vary significantly between the experimental groups.

Table 1

Primers used for the TaqMan® Gene Expression Assays (ThermoFisher).
Gene ID	GenBank accession numbers	Products size (bp)
Actb (β-actin)	Mm02619580_g1	143
Pappa2	Mm01284029_m1	70
Ghrh	Mm00439100_m1	65
Ghih-sst	Mm00436671_m1	86
Ghrhr	Mm01326479_m1	65
Sstr1	Mm00436679_s1	60
Gh	Mm00433590_g1	56
Ghr	Mm00439093_m1	61
Igf1	Mm00439560_m1	77
Igf2	Mm00439564_m1	107
Igf1r	Mm00802831_m1	106
Igfbp3	Mm01187817_m1	78
Igfbp5	Mm00516037_m1	70
Igfals	Mm07307442_s1	63
Stc1	Mm01322191_m1	63

Western blotting analysis

Western blotting was performed as previously described [27]. Briefly, hypothalamus, pituitary and liver samples were homogenized in 500 µL of ice-cold lysis buffer containing Triton X-100, 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 M ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, sodium fluoride (NaF), sodium orthovanadate (NaOV) and protease inhibitors using a tissue-lyser system (Qiagen). After centrifuging at 26,000 g for 30 min at 4ºC, the supernatant was transferred to a new tube. The Bradford method was used to measure the protein concentration of the samples. An amount of 30 µg of each total protein sample was separated on 4–12% polyacrylamide gradient gels. The gels were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) and stained with Ponceau red. Membranes were blocked in TBS-T (50 mM Tris-HCl pH 7.6, 200 mM NaCl, and 0.1% Tween 20) with 2% albumin fraction V from bovine serum (BSA, Roche, Mannheim, Germany) for 1 h at room temperature. The primary antibodies to the proteins of interest (Table 2) were incubated overnight at 4ºC. Mouse adaptin–γ was used as reference protein. After several washes in TBS-T containing 1% Tween 20, an HRP-conjugated anti-rabbit or anti-mouse IgG (H + L) secondary antibody (Promega, Madison, WI, USA), diluted 1:10,000, was added followed by incubation for 1 h at room temperature. After extensive washing in TBS-T, the membranes were incubated for 1 min with the Western Blotting Luminol Reagent kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the specific protein bands were visualized and quantified by chemiluminescence using a ChemiDocTM MP Imaging System (Bio-Rad, Barcelona, Spain). The results are expressed as either the phosphorylated form of target protein/target protein ratios or target protein/adaptin–γ ratios. For more information, see Supplementary Figs. 1–3.

Table 2

Antibodies used for protein expression by Western blotting.
Antigen	MW (kDa)	Immunogen	Manufacturing details	Dilution
Adaptin-γ	104	Mouse adaptin-γ aa. 642–821	BD Biosciences (#610385). Mouse monoclonal antibody	1:2000
Mammalian target of rapamycin (mTOR)	289	Synthetic peptide corresponding to residues surrounding Ser2481 of human mTOR.	Cell Signaling Technology (#2972). Rabbit polyclonal antibody	1:1000
Phospho-mTOR (Ser2448)	289	Synthetic phosphopeptide corresponding to residues surrounding Ser2448 of human mTOR.	Cell Signaling Technology (#2971). Rabbit polyclonal antibody	1:1000
Phosphoinositide 3-kinase or PI3K p85 [19H8]	85	Synthetic peptide corresponding to the sequence of human PI3K p85.	Cell Signaling Technology (#4257). Rabbit monoclonal antibody	1:1000
Phospho-PI3K-p85 (Tyr607)	85	Synthetic peptide corresponding to Human PI 3 Kinase p85 alpha (phospho Y607) conjugated to keyhole limpet haemocyanin.	Abcam (#182651). Rabbit polyclonal antibody	1:1000
AMP-activated protein kinase or AMPKα [D63G4]	62	Synthetic peptide corresponding to residues surrounding Lys40 of human AMPKα.	Cell Signaling Technology (#5832). Rabbit monoclonal antibody	1:1000
Phospho-AMPKα (Thr172) [40H9]	62	Synthetic peptide corresponding to residues surrounding Thr172 of human AMPKα protein.	Cell Signaling Technology (#2535). Rabbit monoclonal antibody	1:1000
Protein kinase B (PKB) or AKT	60	Synthetic peptide corresponding to the carboxy-terminal sequence of mouse AKT.	Cell Signaling Technology (#9272). Rabbit polyclonal antibody	1:1000
Phospho-AKT (Ser473)	60	Synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse AKT.	Cell Signaling Technology (#9271). Rabbit polyclonal antibody	1:1000
Glycogen synthase kinase-3 or GSK-3β [D5C5Z]	46	Recombinant protein specific to the carboxy terminus of human GSK-3β protein.	Cell Signaling Technology (#12456). Rabbit monoclonal antibody	1:1000
Phospho-GSK-3β (Ser9) [D85E12]	46	Synthetic phosphopeptide corresponding to residues surrounding Ser9 of human GSK-3β.	Cell Signaling Technology (#5558). Rabbit monoclonal antibody	1:1000
p44/42 Mitogen-activated protein kinases - MAPK (ERK1/2)	42, 44	Synthetic peptide corresponding to residues near the C-terminus of human p44 MAP kinase.	Cell Signaling Technology (#4695). Rabbit monoclonal antibody	1:1000
Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)	42, 44	Synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.	Cell Signaling Technology (#9101). Rabbit polyclonal antibody	1:1000
Glygogen synthase (GS)	84	Synthetic peptide corresponding to the sequence of human glycogen synthase.	Cell Signaling Technology (#3893). Rabbit polyclonal antibody	1:1000
Phospho-GS (Ser641)	85	Synthetic phosphopeptide corresponding to residues surrounding Ser641 of human liver glycogen synthase.	Cell Signaling Technology (#3891). Rabbit polyclonal antibody	1:1000

Statistical analysis

Data were analyzed using Graph-Pad Prism 7.0 software. Values are expressed as mean ± SEM for each experimental group (n = 8–10 for auxological, hormonal and mRNA analysis; n = 6 for protein analysis). The significance of difference within and between groups was analyzed by two-way ANOVA (sex and genotype as factors) followed by Tukey post-hoc test for multiple comparisons. A p-value less than 0.05 was considered statistically significant.

Effects of Pappa2 deletion on body and bone length

Since previous studies reported that Pappa2 deletion reduces IGF1 bioavailability and affects postnatal growth [4], here we first analyzed the body and bone length of Pappa2^ko/ko mice (Fig. 1). A significant genotype effect on the body length at weaning (PND21) was found (F_1,44 = 15.95, p < 0.001), with ko/ko males having an overall decrease in body length compared to wt/wt males (***p < 0.001; Fig. 1A). At puberty (PND40), the significant effect of genotype on body length remained (F_1,44 = 56.01, p < 0.001), with ko/ko males and females being shorter than their respective wt/wt groups (***p < 0.001; Fig. 1B). We also observed that wt/wt and ko/ko females were shorter than same genotype males (^##/###p < 0.01/0.001; sex effect: F_1,44 = 36.95, p < 0.001; Fig. 1B). In adulthood (8 months of age), there was a significant interaction between genotype and sex on body length (F_1,44 = 4.05, p < 0.05), suggesting that PAPP-A2 deficiency reduced body length differentially in male and female mice with Pappa2 deletion. In addition, effects of genotype (F_1,44 = 90.47, p < 0.001) and sex (F_1,41 = 3.99, p = 0.05) were found on body length in adulthood, with body length in ko/ko males and females being reduced compared to their respective wt/wt groups (***p < 0.001; Fig. 1C), and with wt/wt and ko/ko females being shorter than the respective male groups (^#p < 0.05; Fig. 1C).

In adulthood (8 months of age), the length of long bones (femurs and tibias) was also affected, with a significant interaction between genotype and sex found in femur length (F_1,28 = 6.37, p < 0.05) and femur length relative to body length (F_1,28 = 8.35, p < 0.01), suggesting that PAPP-A2 deficiency reduced femur length differentially in male and female mice with Pappa2 deletion. In addition, effects of genotype (F_1,28 > 27.87, p < 0.001) and sex (F_1,28 > 7.65, p < 0.01) were observed on femur length and femur length relative to body length (Fig. 1D, E), with ko/ko males and females having shorter femurs than their respective wt/wt groups (***p < 0.001), and with wt/wt females having shorter femurs than wt/wt males (^##/###p < 0.01/0.001). No significant interaction was observed on tibia length. A significant effect of genotype was found on tibia length (F_1,28 = 53.45, p < 0.001) and tibia length relative to body length (F_1,28 = 43.52, p < 0.001), with ko/ko males and females having a shorter tibia length than their respective wt/wt groups (***p < 0.001; Fig. 1F, G).

Effects of Pappa2 deletion on circulating and/or pituitary concentrations of IGF1, IGFBP5 and GH

Since bone length deficits could be consequent to hormonal alterations, we assessed plasma and/or pituitary concentrations of IGF1, IGFBP5 and GH in Pappa2^ko/ko mice (Fig. 2). A significant interaction between genotype and sex was found in the circulating levels of IGF-1 (F_1,28 = 17.20, p < 0.001) and IGFBP5 (F_1,28 = 9.22, p < 0.01), suggesting that the plasma levels of both IGF-1 and IGFBP5 were differentially increased in male and female mice with Pappa2 deletion (Figs. 2A, B). A genotype effect was found on the circulating levels of IGF-1 (F_1,28 = 29.14, p < 0.0001), with an overall increase in IGF-1 levels in ko/ko females compared to wt/wt females (***p < 0.001; Fig. 2A). We also found sex effects on the circulating levels of IGF1 (F_1,28 = 25.85, p < 0.0001) and IGFBP5 (F_1,28 = 11.65; p < 0.01), with the ko/ko female group having significantly more IGF1 and IGFBP5 that the ko/ko male group (^###p < 0.001; Figs. 2A, B). No effects or interaction were found on the plasma levels of GH (Fig. 2C).

Since GH is secreted in a pulsatile fashion and a single plasma measurement does not necessarily reflect its total integrated levels, we also analyzed GH concentrations in Pappa2^ko/ko mice at the pituitary level (Fig. 2D). An effect of genotype was found on pituitary GH content (F_1,28 = 3.99, p < 0.05), which was most likely due to increased levels of GH in the pituitary gland of the ko/ko males and females (Figs. 2D).

Effects of Pappa2 deletion on gene expression of the hypothalamic IGF1 system

We analyze whether Pappa2 insufficiency affects gene expression of the main components of the IGF1 system in the hypothalamus (Fig. 3). An effect of genotype was found on the mRNA levels of Pappa2 (F_1,28 = 123.2, p < 0.001), with an overall decrease in Pappa2 mRNA in ko/ko males and females compared to their respective wt/wt groups (***p < 0.001; Fig. 3A). There was a sex effect on the mRNA levels of Ghrh (F_1,28 = 6.649, p < 0.05), with females having lower Ghrh mRNA levels than males (Fig. 3B). No interactions or main effects were found in Ghih expression (Fig. 3C).

No interactions or main effects were found in Igf1 expression (Fig. 3D), but a sex effect was found on the mRNA levels of Igf1r (F_1,28 = 7.547, p < 0.05), with females having lower Igf1r mRNA levels compared to males (Fig. 3E). No interactions or main effects were found in Igfbp3 expression (Fig. 3F). An effect of genotype (F_1,28 = 5.681, p < 0.05) was found on the mRNA levels of Igfbp5, with ko/ko males having higher levels compared to wt/wt males (*p < 0.05; Fig. 3G). There was a sex effect on the mRNA levels of Igfals (F_1,28 = 4.445, p < 0.05), with females having higher mRNA levels compared to males (Fig. 3H). No main effects or interactions between factors were found in the Stc1 mRNA levels (Fig. 3I).

Effects of Pappa2 deletion on gene expression of the pituitary IGF1 system

We also analyzed the gene expression of the main components of the IGF1 system in the pituitary gland of Pappa2^ko/ko mice (Fig. 4). A significant interaction between sex and genotype (F_1,28 = 34.22; p < 0.001), and main effects of sex (F_1,28 = 48.96; p < 0.001) and genotype (F_1,28 = 219.0, p < 0.001) were found on the mRNA levels of Pappa2, with an overall decrease in ko/ko male and female mice compared to respective wt/wt mice (***p < 0.001; Fig. 4A), and lower levels of Pappa2 mRNA in wt/wt females compared to wt/wt males (^###p < 0.001; Fig. 4A). An interaction between sex and genotype (F_1,28 > 8.177, p < 0.01) and a sex effect (F_1,28 > 31.88, p < 0.001) were found on the mRNA levels of both Gh and Ghrhr. Multiple comparison tests showed lower levels of Gh and Ghrh in wt/wt females than wt/wt males (^###p < 0.001; Figs. 4B, C). We also found lower Ghrh levels in the pituitary gland of ko/ko males compared to wt/wt males (*p < 0.05; Fig. 4C).

A significant interaction between sex and genotype (F_1,28 = 17.01; p < 0.001), and main effects of sex (F_1,28 = 23.47; p < 0.001) and genotype (F_1,28 = 23.75; p < 0.001) were found on the mRNA levels of Igf1. Multiple comparison tests indicated overall higher levels if Igf1 mRNA in ko/ko females compared to wt/wt females and ko/ko males (***^/###p < 0.001; Fig. 4D). No interaction or effects between factors were found in the pituitary mRNA levels of Igf1r, Igfbp3, Igfbp5, Igfals or Stc1 (Figs. 4E-I).

Effects of Pappa2 deletion on gene expression of the liver IGF1 system

Since changes in PAPP-A2 activity can also affect the liver, we analyzed the gene expression of the main components of the IGF1 system in the liver of Pappa2^ko/ko mice (Fig. 5). Main effects of sex (F_1,28 = 8.932, p < 0.01) and genotype (F_1,28 = 13.87, p < 0.01) were found on the mRNA levels of Ghr, with an overall increase in Ghr mRNA in ko/ko male and female groups compared to their respective wt/wt groups (*p < 0.05; Fig. 5A).

A significant interaction between sex and genotype was found in the mRNA levels of Igf1 (F_1,23 = 9.216, p < 0.01), suggesting that the liver mRNA levels of Igf1 were differentially altered in male and female mice with Pappa2 deletion (Figs. 5B). We also found a genotype effect on the mRNA levels of Igf1 (F_1,28 = 16.38, p < 0.001). Multiple comparison analysis indicated that ko/ko females showed higher mRNA levels of Igf1 than wt/wt females (***p < 0.001) and ko/ko males (^##p < 0.01; Fig. 5B). No interaction or effects were found on the mRNA levels of Ifg1r or Igf2 (Figs. 5C, D).

An interaction between sex and genotype (F_1,28 = 5.518, p < 0.05), and an effect of genotype (F_1,28 = 6.400, p < 0.05) on the mRNA levels of Igfbp3 were found, with ko/ko females having higher Igfbp3 expression compared to wt/wt females (*p < 0.05; Fig. 5E). An effect of sex was found on the mRNA levels of Igfbp5 (F_1,28 = 6.59; p < 0.05), with females having higher expression (Fig. 5F). An effect of genotype was found on the mRNA levels of Igfals (F_1,28 = 10.52; p < 0.01), with an increase in Igfals expression in ko/ko females compared to wt/wt females (*p < 0.05; Fig. 5G). A genotype effect was also found in the mRNA level of Stc1 (F_1,28 = 11.00, p < 0.01), with ko/ko males and females expressing significantly more Stc1 than their respective wt/wt groups (*p < 0.05; Fig. 5H). Pappa2 mRNA was undetected in the liver of all experimental groups analyzed.

Effects of Pappa2 deletion on IGF1 signaling pathways in the hypothalamus

To further understand how PAPP-A2 deficiency affects hypothalamic regulatory systems, we analyzed IGF1 signaling pathways in this brain area (Fig. 6). No interaction or effects were found on the phosphorylated form (Tyr607) of PI3K (Fig. 6A). A significant interaction between sex and genotype was found in the protein levels of AKT when it was phosphorylated (p) on serine 473 (F_1,20 = 22.25, p < 0.001), mTOR phosphorylated on serine 2448 (F_1,20 = 57.51, p < 0.001), GSK3β phosphorylated on serine 9 (F_1,20 = 4.57; p < 0.05), ERK1 phosphorylated on threonine 202 (F_1,20 = 15.54, p < 0.001), and ERK2 phosphorylated on tyrosine 204 (F_1,20 = 4.79, p < 0.05), suggesting that the hypothalamic levels of p(Ser473)-AKT, p(Ser2448)-mTOR, p(Ser9)-GSK3β, p(Thr202)-ERK1 and p(Tyr204)-ERK2 were differentially altered in male and female mice with Pappa2 deletion (Figs. 6B-F). We also found genotype effects on the hypothalamic levels of p(Ser473)-AKT (F_1,20 = 7.024; p < 0.05) and p(Thr202)-ERK1 (F_1,20 = 5.29, p < 0.05). Effects of sex were found on the protein levels of p(Ser473)-AKT (F_1,20 = 5.22, p < 0.05), p(Ser9)-GSK3β (F_1,20 = 7.47, p < 0.05) and p(Thr202)-ERK1 (F_1,20 = 9.029, p < 0.01). No interaction or effects were found on the protein levels of p(Thr172)-AMPKα or p(Ser641)-GS (Figs. 6G, H).

Multiple comparison analysis indicated that protein levels of p(Ser473)-AKT, p(Ser2448)-mTOR and p(Thr202)-ERK1 were higher in the hypothalamus of ko/ko females compared to wt/wt females (**^/***p < 0.01/0.001) and ko/ko males (^##/###p < 0.01/0.001; Figs. 6B, C, E). However, ko/ko males had lower p(Ser2448)-mTOR than wt/wt males (**p < 0.01; Fig. 6C). Regarding sex differences, ko/ko female had higher levels of p(Ser9)-GSK3β and p(Tyr204)-ERK2 than ko/ko males (^#/##p < 0.05/0.01; Figs. 6D, F), whereas wt/wt females had lower p(Ser2448)-mTOR wt/wt males (^##p < 0.01; Figs. 6C). Representative immunoblots are shown in Fig. 6I.

Effects of Pappa2 deletion on IGF1 signaling pathways in the pituitary gland

We also analyzed IGF1 signaling pathways in the pituitary gland (Fig. 7). An interaction between sex and genotype (F_1,20 = 5.9; p < 0.05), and effects of sex (F_1,20 = 11.08, p < 0.01) and genotype (F_1,20 = 7.42, p < 0.05) were only found in the protein levels of p(Ser9)-GSK3β. Multiple comparison analysis indicated that protein levels of p(Ser9)-GSK3β were higher in ko/ko males compared to wt/wt males and ko/ko females (**^/##p < 0.01; Fig. 7A). No interaction or effects were found on the protein levels of p(Thr172)-AMPKα, p(Thr202)-ERK1 or p(Tyr204)-ERK2 (Figs. 7B-D). Representative immunoblots are shown in Fig. 7E.

Effects of Pappa2 deletion on IGF1 signaling pathways in the liver

The IGF1 signaling pathways were also analyzed in the liver of Pappa2^ko/ko mice (Fig. 8). An interaction between sex and genotype was observed when protein levels of p(Tyr607)-PI3K were analyzed (F_1,20 = 6.55, p < 0.05), suggesting that the protein levels of p(Tyr607)-PI3K were differentially altered in the liver of male and female mice with Pappa2 deletion (Fig. 8A). Effects of genotype were found on the protein levels of p(Tyr607)-PI3K (F_1,20 = 10.93, p < 0.01) and p(Thr172)-AMPKα (F_1,20 = 6.264, p < 0.05), with higher protein levels in the liver of ko/ko females compared to wt/wt females (*/**p < 0.05/0.01; Figs. 8A, G). Effects of sex were found on the protein levels of p(Ser2448)-mTOR (F_1,20 = 4.19, p = 0.05) and p(Thr172)-AMPKα (F_1,20 = 8.185, p < 0.05), with females having lower protein levels than males (^#p < 0.05; Figs. 8C, G). No interaction or effects on protein levels of p(Ser473)-AKT, p(Ser9)-GSK3β, p(Thr202)-ERK1, p(Tyr204)-ERK2 or p(Ser641)-GS were found (Figs. 8B, D, E, F, H). Representative immunoblots are shown in Fig. 8I.

In the present study, we describe the expression of the main components of IGF1 ternary complexes and IGF1 intracellular signaling pathways in the hypothalamus, pituitary gland and liver, key tissues involved in the physiological control of growth, in an animal model of Pappa2 deficiency. Moreover, special emphasis was placed on the differential responses of males and females to this genetic alteration. It is known that PAPP-A2 is a key regulator of IGF1 bioavailability and that IGF1, acting as an autocrine, endocrine and paracrine factor, is responsible for promoting efficient growth, development and maintenance at all physiological stages. As growth and the GH/IGF system are sexually dimorphic in rodents [28] and human in an age-specific manner [12], analyzing sex-specific IGF1 system alterations due to the lack of PAPP-A2 proteolytic activity can provide relevant information about possible differences in the implications of PAPP-A2 in growth physiology between males and females. In addition, it may also contribute to clarify the physiopathological mechanisms and signaling pathways underlying the alterations in IGF1 bioavailability observed in PAPP-A2 deficient patients with progressive postnatal growth retardation.

Homozygous Pappa2^ko/ko mice recapitulate several features of the phenotype described in patients with PAPP-A2 mutations [1, 3, 4, 11], especially regarding auxology (short stature, thin long bones, decreased bone mineral density), hormone levels (elevated serum concentrations of IGF1, IGFBP3, IGFBP5 and IGFALS) and metabolic status (disturbance in carbohydrate metabolism). Pappa2 deficient mice have growth retardation from the neonatal period to adulthood, with Pappa2^ko/ko mice having reduced body and bone (femur and tibia) length throughout lifespan [20,21,23], which was also found here. In addition, the IGF1 system was described to be altered at the local level in bone of these mice, which probably contributes to not only reduced bone growth but also to the alterations in bone structure (crystallographic parameters), composition (mineral-to-organic matrix ratio, carbonate substitution, collagen maturity) and remodeling (osteopontin, osteocalcin, alpha-1 type 1 collagen) in a sex-dependent manner [16,23].

Here mice lacking Pappa2 also present differences in plasma concentrations of the main components of IGF1 ternary/binary complexes (e.g., IGF1, IGFBP5), similar to PAPP-A2 deficient patients [4], with a difference between the sexes shown in mice. The absence of Pappa2 caused an increase in plasma levels of IGF1, especially in female mice. Pappa2^ko/ko females also had significant increases in plasma levels of IGFBP5, whose binding to IGF1 is directly controlled by the proteolytic activity of PAPP-A2 [7, 8]. Total IGF1 levels were found to have a similar plasma profile, suggesting that part of IGF1 is bound in ternary and/or binary complexes in conjunction with IGFBP5. Therefore, one would expect reduced IGF1 actions through IGF1R in target tissues. Low plasma levels of bioactive IGF1 would reduce its negative feedback actions on the production and secretion of GH by somatotropic cells of the anterior pituitary gland. In both Pappa2^ko/ko males and females GH concentrations were increased in the pituitary gland, an effect that could result in more liver production of IGF1 and IGFBP5 [29]. In our animal model, increased GH secretion is supported by higher mRNA levels of GH targets, such as Ghr, Igf1, Igfbp3, Igfals and Stc1, at least in the liver of Pappa2^ko/ko females. Thus, stimulation of IGF1 expression and secretion could be one of the main responses to overcome the lack of IGF1R signaling and to palliate the reduced activation of mechanisms controlling growth physiology in PAPP-A2 deficiency [1,8,23].

Although low IGF1 bioavailability would increase GH synthesis, as observed here, this increase is not reflected in the plasma of animals lacking Pappa2, as previously reported in patients with PAPP-A2 deficiency [1]. However, as GH is secreted in a pulsatile fashion, a single plasma measurement does not necessarily reflect its total integrated levels, nor the efficiency of the pulsatile pattern to promote growth. Several studies reported that there is not always a perfect correlation between IGF1 and GH actions, suggesting that IGF1 production depends on additional factors that modulate this axis [30, 31, 32]. In agreement, mice with liver IGF1 deficiency (LID) have reduced circulating total IGF1 levels and a consequent increase in plasma GH levels, whereas mice with Igfals gene deletion (ALSko) have reduced levels of circulating total IGF1 with normal GH levels [30]. Only double-knockout mice (LID + ALSko) were described to have a marked increase in free IGF1 levels [30].

Sex-based modulation of the IGF1 system at the tissue level has not been described to date; thus, we analyzed the gene expression of the main components of the IGF1 system in the hypothalamus, pituitary gland and liver of male and female Pappa2^ko/ko mice. As expected, Pappa2 mRNA levels were dramatically reduced in the hypothalamus and pituitary gland of Pappa2^ko/ko male and female mice, and no Pappa2 expression was detected in their liver. Significantly attenuated proteolytic activity of PAPP-A2 in the hypothalamus and pituitary gland (and probably in the liver) would most likely reduce local IGF1 bioavailability and increase circulating IGF1 half-life, probably due to increased expression of components of IGF ternary/binary complexes. Following a comprehensive analysis of the mRNA levels of IGF1 system components in the hypothalamus-pituitary-liver axis, animals lacking Pappa2 presented changes in the gene expression of Igf1, Igfbp3, Igfbp5, Igfals, Stc1, Ghr, with some changes being sex-dependent (Pappa2, Gh, Ghrhr, Igf1, Igfbp3). In fact, there are marked sex differences in the mRNA levels of IGF1 system components (Pappa2, Ghrh, Ghrhr, Gh, Ghr, Igf1, Igf1r, Igfbp5, Igfals) in different tissues in this animal model. In Pappa2^ko/ko mice increased mRNA levels of Igfbp5 were found in the hypothalamus, Igf1 in the pituitary gland, and Igf1, Igfbp3, Igfals and Stc1 in the liver. These results, together with increased circulating concentrations of IGF1 and IGFBP5, agree with an upregulated expression of key components of the IGF1 ternary/binary complexes that probably result in lower local levels of bioactive IGF1 in the hypothalamus, pituitary gland and liver. Although animals lacking Pappa2 show a dysregulated IGF1 system, the gene expression of hypothalamic hormones involved in pituitary GH production, such as Ghrh and Ghih, is not particularly affected by genotype. These results suggest that the increased GH levels in the pituitary gland could be a direct consequence of decreased negative feedback of IGF1 at this level. It should also be noted that previous studies suggest a central and peripheral role of IGFBP5 in energy metabolism, with animals lacking Igfbp5 and fed a fatty diet showing glucose intolerance and increased body weight and adiposity [33]. In fact, Igfbp5 expression was specifically increased in the hypothalamus of male mice fed a high-fat diet without affecting other members of the IGF system [34], and IGFBP5 was altered in the hypothalamus of Pappa2^ko/ko mice of both sexes.

The increased Ghr mRNA levels in the liver of Pappa2^ko/ko male and female mice (genotype effect) substantiate an increase in GH-stimulated IGF1 expression in the liver [29], although this GH increase is only reflected in the pituitary gland, and not in plasma, of animals lacking Pappa2. Sex-specific effects of Pappa2 deficiency on the gene expression of IGF1 system components are represented by: 1) a specific increase of Igfbp5 in the hypothalamus of Pappa2^ko/ko males; 2) specific increases of Gh and Igf1 in the pituitary gland of Pappa2^ko/ko females; 3) a decrease of Ghrhr in the pituitary gland of Pappa2^ko/ko males; and 4) increases of Igf1, Igfbp3 and Igfals in the liver of Pappa2^ko/ko females. The female-specific increase in the expression of components of the IGF1 system in liver suggests that the increased synthesis of IGF1, most likely stimulated by GHR activation in the liver (increased expression of pituitary GH and liver Ghr) is mainly sequestered in IGF ternary/binary complexes of Pappa2^ko/ko females, also supported by the increased levels of circulating levels of IGFBP5. It is plausible to assume that this may result in reduced IGF1 bioavailability in target tissues. Therefore, the hypothalamus, pituitary gland and liver may be exposed to reduced IGF1-IGF1R interactions that could involve subsequent alteration of IGF1 intracellular signaling pathways in a sex-specific manner. However, in Pappa2^ko/ko females there was no change in the expression of the hypothalamic factors analyzed here. Moreover, the intracellular signaling pathways analyzed were either unaffected or increased in these females. These data indicate that the lack of Pappa2 may have limited effects on the IGF system at the level of the hypothalamus.

Longitudinal growth and somatic maturation are mainly regulated by IGF1 binding to IGF1R and the subsequent IGF1R activation of main intracellular signaling pathways in target tissues [29, 35, 36, 37]. To determine sex differences in putative IGF1 signaling pathway dysregulation in response to Pappa2 deficiency, we analyzed key intracellular pathways, including PI3K, AKT, AMPKα, ERK1/2, mTOR and GSK3β, among others, in the hypothalamus-pituitary-liver axis of Pappa2^ko/ko male and female mice. Sex-specific effects of Pappa2 deficiency on these signaling pathways included: 1) a decrease p(Ser2448)-mTOR in the hypothalamus of Pappa2^ko/ko males, and increases of p(Ser2448)-mTOR, p(Thr202/Tyr204)-ERK1/2, p(Ser473)-AKT and p(Ser9)-GSK3β in the hypothalamus of Pappa2^ko/ko females; 2) an increase of p(Ser9)-GSK3β (a tendency of p(Thr202/Tyr204)-ERK1/2) in the pituitary gland of Pappa2^ko/ko males; and 3) increases of p(Tyr607)-PI3K and p(Thr172)-AMPKα in the liver of Pappa2^ko/ko females (Fig. 9).

There are key signaling pathways via RAS/MEK/ERK and PI3K/AKT/mTOR that are activated following cellular activation of IGF1R, and mainly serve the function of regulating unrestricted cell proliferation and decreased sensitivity to apoptotic mechanisms that involve changes in glucose and lipid metabolism and protein synthesis in target tissues responsible for promoting growth, development, and maintenance at all physiological stages [38, 39, 40]. AKT, a principal target of PI3K signaling for IGF1, regulates glucose metabolism (glucose transportation, glycogen synthesis and gluconeogenesis inhibition) via GSK3 phosphorylation/inhibition and mTOR activation [38, 40, 41]. Increased phosphorylated levels of AKT, GSK3β and mTOR were specifically found in the hypothalamus of Pappa2^ko/ko females, and this may suggest a close regulatory control of glucose metabolism and protein synthesis in the female hypothalamus. Local activation of the PI3K/AKT signaling pathway, via the subsequent mTOR activation, has a key role in protein synthesis to coordinate neural cell proliferation, neuronal differentiation and polarization and axonal growth during brain development, as well as axonal regeneration after injury, effects that are promoted by the suppression of GSK3β (Ser9)-phosphorylation/inactivation, and its dysregulation is associated with many psychiatric and neurodegenerative diseases [42, 43]. Regarding our results, overexpression of GSK3β (Ser9)-phosphorylation in the hypothalamus of Pappa2^ko/ko female mice could promote premature neuronal differentiation and prevent axon formation, effects that can be dramatic during brain development. Our results also suggest that higher ERK1/2 phosphorylation (also known as p44 and p42 MAPK) is probably inducing cell proliferation in the hypothalamus of Pappa2^ko/ko females (Fig. 9). Together these results could indicate increased cell turnover, or even increased metabolism; hypotheses that should be analyzed in future studies.

It is also important to focus on the hypothalamic AMPK signaling pathway via mTOR, a primary oxygen and nutrient sensing pathway that plays a key role in the central modulation of glucose and amino acid utilization and signaling through IGF1, an effect that may occur in a sex-specific manner [44, 45]. Under stressful maternal environments and elevated cortisol levels, mTOR signaling is affected and this effect may explain differences in male and female growth and development in these circumstances [45]. Indeed, AMPK-mTOR signaling is also necessary for bone development through promotion of chondrocyte proliferation and hypertrophy [46]. Our results suggest that sex-specific effects of Pappa2 deficiency on the hypothalamic expression of phosphorylated mTOR, that is decreased levels in Pappa2^ko/ko males and increased levels in Pappa2^ko/ko females (Fig. 9), could result in changes in the hypothalamic control of growth physiology and maintenance in a sex-dependent manner.

In pituitary somatotropic cells, ERK signaling becomes activated by GHRH and inhibited by somatostatin, resulting in increased proliferative and non-proliferative responses, respectively [47]. In addition, activation of ERK signaling is required for GH production, while inhibition of PKA/CREB signaling pathway via GSK3β dephosphorylation/activation is necessary to suppress GH synthesis in somatotrophs [47, 48, 49]. Despite no evidence of an AMPK role in hypothalamic control of somatotropic function, AMPK activation was followed by a reduction of GH storage and release in the pituitary gland, an effect that may be inhibited by ERK1/2 phosphorylation and enhanced by the negative feedback regulatory action of IGF1 on the pituitary somatotropic cells [18]. These studies support the fact that the genotype effect due to increased levels of phosphorylated GSK3β (and a tendency in phosphorylated ERK1/2) agree with increased GH secretion in the pituitary gland at least in Pappa2^ko/ko males, as was described above, whereas normal GSK3 expression in the pituitary gland of Pappa2^ko/ko females may result in a limited effect on GH production, suggesting that different mechanisms could be involved between sexes (Fig. 9).

Accumulated evidence indicates that PI3K/AKT pathway activation and transcription factor-induced synthesis and release of IGF1 in the liver are required for normal longitudinal growth before and during puberty, body composition (increasing body mass and decreasing body fat) and protein, lipid, carbohydrate and mineral metabolism [17]. IGF1 elicits transient insulin-like activation of the PI3K p85 subunit, leading to AKT phosphorylation and GSK3 inactivation, and thereby facilitating glycogen synthesis, fatty acid synthesis for storage, cell apoptosis protection and cell cycle progression [50]. In addition, AMPK activity has the potential to interfere with GH-induced IGF1-mediated cellular anabolic events by modulating PI3K/AKT pathways in the liver [18]. Activated AMPK switches off glycogen and protein synthesis and cell cycle progression, leading to adaptive changes in response to caloric restriction and negative balance, including insulin sensitivity, through inhibiting the mTOR response [18]. A controversial study treating HepG2 with free fatty acids and using high-fat diet models, associated IGFBP5 overexpression that may result in low IGF1 bioavailability with increased glucose uptake and glycogen synthesis through activating both IRS1/AKT and AMPK pathways [51]. Our results suggest that increased phosphorylated levels of PI3K and AMPK, specifically observed in the liver of Pappa2^ko/ko female mice, may explain higher production of IGF1 and IGFBP5, and key components of IGF1 ternary/binary complexes (Igf1, Igfbp3 and Igfals), in addition to blocking anabolic events leading to a negative energy balance [4].

Perspective And Sifnificance

This study presents a systematic description of the sex-specific changes found in the IGF1 system and IGF1 signaling pathways through the hypothalamus-pituitary-liver axis in a Pappa2-deficient animal model with growth retardation. Reduction in body and bone length of Pappa2^ko/ko mice and higher expression of specific components of IGF1 binary/ternary complexes in a female-specific manner (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. Indeed, sex-specific alterations of regulators of IGF1 signaling pathways (AKT, mTOR, GSK3β and ERK1/2 phosphorylation in the female hypothalamus, GSK3β and ERK1/2 phosphorylation in the male pituitary gland, and PI3K and AMPKα phosphorylation in the female liver) could be relevant to explain growth impairment through a dysregulated IGF1 system due to PAPP-A2 deficiency. These results shed light on the involvement of PAPP-A2 in sex-based growth physiology, and contribute to elucidate the molecular mechanisms underlying IGF1 signaling disruption in PAPP-A2 deficient patients with physiological and metabolic derangements related to progressive postnatal growth retardation. Further studies are necessary to determine how post-pubertal sex steroids, which are clearly involved in growth promotion and modulation of the GH-IGF system, interact with the lack of Pappa2.

Ethics approval and consent to participate

Ethics approval and consent to participate involving human participants, human data or human tissue are not applicable. This manuscript involves the use of animal data and tissue. All animal procedures were conducted in strict adherence to ARRIVE guidelines and the principles of laboratory animal care (National Research Council, Neuroscience CoGftUoAi, Research B, 2003) following the European Community Council Directive (86/609/EEC). All animal procedures were approved by the Ethical Committee of the University of Málaga (Exp. Nº: 46-2019-A).

Consent for publication

Not applicable

Acknowledgements

We would like to acknowledge professor Julian Christians for providing the first colony of Pappa2^ko/ko mice.

Funding

This study was supported by the Spanish Ministry of Science and Innovation with the help of European Regional Development Funds-European Union (ERDF-EU) (PI19/00166 and PI19/00343), Ministerio de Economía y Competitividad (BFU 2017-82565-C2-1-R) and the Network Center for Biomedical Research on Obesity and Nutrition (CIBEROBN) and Instituto de Salud Carlos III (ISCIII). P.R. (CP19/00068) holds a ‘’Miguel Servet I” research contract from the National System of Health, ERDF-EU-ISCIII. A.J.L.-G. (IFI18/00042) holds an “iPFIS” predoctoral contract from the National System of Health, ERDF-EU-ISCIII.

Contributions

Conceptualization, J.A., J.S. and P.R.; resources, J.A. and J.S.; methodology, J.A.N., A.J.L.-G., M.M.F.A. and L.R.; formal analysis, J.A.N., M. dC., J.S. and P.R.; investigation, M.M.F.A., J.S. and P.R.; data curation, A.J.L.-G., J.S. and P.R.; writing—original draft preparation, J.S. and P.R.; writing—review and editing, F.R.F., V.B., J.A.C. and J.A.; supervision, J.S. and P.R.; project administration, J.A. and J.S.; funding acquisition, J.A.C., J.A. and J.S. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

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.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Supplementary figures 1-3. Additional data of original immunoblots.

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Reviewers invited by journal
03 Jul, 2023
Reviewers agreed at journal
25 Jan, 2023
First submitted to journal
18 Jan, 2023

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Sex-based differences in IGF1 signaling pathways in response to PAPP-A2 deficiency

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Abstract

Background.

Methods.

Results.

Conclusions.

Figures

Background

Methods

Ethics statement

Animals

Sample collection

Hormone analyses

RNA isolation and RT-qPCR analysis

Western blotting analysis

Statistical analysis

Results

Discussion

Perspective And Sifnificance

Declarations

References

Supplementary Files

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Version 1