Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11ß-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11ß enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11ß-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Genetic loss of proadrenomedullin N-terminal 20 peptide (PAMP) in mice is compatible with survival.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are small peptides derived from a common precursor, pre-proadrenomedullin. Although AM and PAMP share hypotensive effects in the cardiovascular system, the peptides also exert diverse and distinct effects on endocrine physiology, innate immunity, cytoskeletal biology and receptor signaling pathways. Tremendous knowledge has been gleaned from the study of several genetic animal models of AM deletion or overexpression, some of which also simultaneously delete the coding region for PAMP peptide. However, deletion of PAMP without concurrent deletion of AM in an animal model is not currently available for the study of PAMP function. Here, we present the generation of AdmΔPAMP/ΔPAMP and AdmΔPAMP/- mice, which lack the coding sequence for PAMP while preserving the coding sequence for AM. AdmΔPAMP/ΔPAMP mice survive to adulthood without any obvious abnormalities and are fertile, though AdmΔPAMP/- females have small litters. Interestingly, these animals express lower levels of Adm mRNA and AM peptide than wild type animals, but these levels are still compatible with survival. Importantly, despite reduced levels, the spatiotemporal expression of AM peptide within the hearts of AdmΔPAMP/- mice remains similar to wild type animals. AdmΔPAMP/ΔPAMP mice are now a publicly available tool for future investigations of PAMP function.

Calcitonin-Receptor-Like Receptor Signaling Governs Intestinal Lymphatic Innervation and Lipid Uptake.

The absorption of dietary fat requires complex neuroendocrine-mediated regulation of chylomicron trafficking through enterocytes and intestinal lymphatic vessels. Calcitonin-receptor-like receptor (Calcrl) is a G protein-coupled receptor that can bind either a lymphangiogenic ligand adrenomedullin, with coreceptor RAMP2, or the neuropeptide CGRP, with coreceptor RAMP1. The extent to which this common GPCR controls lipid absorption via lymphatics or enteric innervation remains unclear. We used conditional and inducible genetic deletion of Calcrl in lymphatics to elucidate the pathophysiological consequences of this receptor pathway under conditions of high-fat diet. Inefficient absorption of dietary fat coupled with altered lymphatic endothelial junctions in Calcrl fl/fl /Prox1-CreER T2 mice results in excessive, transcellular lipid accumulation and abnormal enterocyte chylomicron processing and failure to gain weight. Interestingly, Calcrl fl/fl /Prox1-CreER T2 animals show reduced and disorganized mucosal and submucosal innervation. Consistently, mice with genetic loss of the CGRP coreceptor RAMP1 also displayed mucosal and submucosal innervation deficits, substantiating the CGRP-biased function of Calcrl in the neurolymphocrine axis. Thus, the common Calcrl receptor is a critical regulator of lipid absorption through its cell-specific functions in neurolymphocrine crosstalk.

Lymphatic deletion of calcitonin receptor-like receptor exacerbates intestinal inflammation.

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor-like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrlfl/fl/Prox1-CreERT2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrlfl/fl/Prox1-CreERT2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.