Fig 2: Model of PAPP‐A effects during normal tissue homeostasis and aging. This manuscript demonstrates that PAPP‐A is expressed by MSCs and potentiates IGF signaling in those cells, increasing their activity and/or proliferation and promoting normal tissue function such as ECM production, myeloid cell production, and bone marrow osteogenesis and adipogenesis. However, with age, PAPP‐A‐mediated IGF stimulation in MSCs may become maladaptive, leading to aberrant collagen/ECM deposition that drives fibrosis‐related pathology, and/or MSC functional exhaustion that drives tissue dysfunction. We hypothesize that restraining MSC activity and thereby delaying these fates is the mechanism by which PAPP‐A inhibition extends healthspan and lifespan. Figure created with biorender.com

Fig 3: Model for IGF1-mediated Leptomeningeal Metastasis.In our proposed model, bioavailability of IGF1 is highest at the leptomeningeal surface, where IGF1 can be locally liberated by IGFBP3-cleaving proteases. Metalloproteases including MMP-9 are known to be expressed not only in the brain parenchyma under physiological or diseased states but also by the uninjured or injured leptomeninges484950 and tPA is known to be expressed from the both meninges as well as the vertebral artery5152. Both TIMP1-MMP-IGFBP3-IGF and SerpinE1/PAI-1-tPA-IGFBP3-IGF axes have been previously described in other model systems303134.

Fig 4: PI3K activates MAPK via the p110α-Ras interaction.(A) In p110αRBD/RBD MEF cells, the loss of the p110α-Ras interaction not only prevented FGF from inducing AKT phosphorylation but also down-regulated the levels of pERK and active Ras (Ras-GTP). One-way ANOVA test for statistical significance (n = 2). (B) Deletion of p110α and p110β in MEF cells disrupted IGF-induced AKT and ERK phosphorylation. One-way ANOVA test for statistical significance (n = 2). (C) pERK staining was down-regulated in p110RBD glands and sharply reduced in p110CKO mutants. Expression of the constitutively active Mek1DD allele led to partial recovery of lacrimal gland budding and pERK staining in p110CKO;Mek1DD mutants. One-way ANOVA test (n = 5 for the control and n = 3 for the rest). (D) In comparison to control and Mek1DD pups, p110RBD mutants mostly displayed residual glands at birth, whereas p110RBD;Mek1DD animals showed more extensively branched glands. Similarly, lacrimal gland induction was observed in p110CKO;Mek1DD mutants but not in p110CKO mutants.

Fig 5: Suppression of EGF signaling by PI3K and MAPK is necessary for lacrimal gland budding.(A) Egfr expression obtained by RNA-seq analysis was elevated in p110RBD and p110CKO mutants. FPKM, Fragments Per Kilobase of transcript per Million. (B) In control lacrimal glands, Egfr was expressed in the stalk region but excluded from the bud. This distinction was lost in p110RBD lacrimal glands. In p110CKO, Fgfr2CKO, ErkCKO, and RaptorCKO mutants, Egfr was uniformly expressed in the conjunctiva. (C) The explant assay was performed with the eye tissue dissected from E13.5 Le-Cre embryos that expressed GFP. After cultured on the floating membrane in the presence or absence of EGF, the eye rudiment was evaluated for formation of the GFP+ lacrimal gland bud. (D) In 2 days of culture, the lacrimal gland budding was inhibited by EGF (100 ng/ml) but not bovine serum albumin (BSA). (E) Quantification of lacrimal gland budding. Fisher’s exact test, P = 0.0006. (F) Model of PI3K-MAPK interaction in lacrimal gland development. The p110 subunit of PI3K is activated by both p85-mediated IGF signaling and Ras-mediated FGF signaling. Conversely, the p110α-Ras interaction also activates Ras to stimulate MAPK signaling, which cooperates with AKT to control mTOR activity. We propose that these signaling pathways may suppress Egfr expression (dashed lines), allowing the budding of the lacrimal gland from the conjunctiva.