Gene expression changes contribute to stemness and therapy resistance of relapsed acute myeloid leukemia: roles of SOCS2, CALCRL, MTSS1, and KDM6A.

Relapse is associated with therapy resistance and is a major cause of death in acute myeloid leukemia (AML). It is thought to result from the accretion of therapy-refractory leukemic stem cells. Genetic and transcriptional changes that are recurrently gained at relapse are likely to contribute to the increased stemness and decreased therapy responsiveness at this disease stage. Despite the recent approval of several targeted drugs, chemotherapy with cytosine arabinoside and anthracyclines is still the mainstay of AML therapy. Accordingly, a number of studies have investigated genetic and gene expression changes between diagnosis and relapse of patients subjected to such treatment. Genetic alterations recurrently acquired at relapse were identified, but were restricted to small proportions of patients, and their functional characterization is still largely pending. In contrast, the expression of a substantial number of genes was altered consistently between diagnosis and recurrence of AML. Recent studies corroborated the roles of the upregulation of SOCS2 and CALCRL and of the downregulation of MTSS1 and KDM6A in therapy resistance and/or stemness of AML. These findings spur the assumption that functional investigations of genes consistently altered at recurrence of AML have the potential to promote the development of novel targeted drugs that may help to improve the outcome of this currently often fatal disease.

Potentiated adrenomedullin-induced vasorelaxation during hypoxia in organ cultured porcine coronary arteries.

This study describes the effect of variable oxygen supply on relaxing responses induced by α-calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) on isolated pig coronary arteries in vitro. Organ culture during normoxia (21% of O2) and hypoxia (5% of O2) induced a significant leftward shift of the AM concentration-response curves compared with fresh vessels altering the pEC50 values from 6.9 ± 0.04 to 8.0 ± 0.04, whereas the potency (pEC50) of αCGRP was attenuated from 8.8 ± 0.04 to 7.6 ± 0.04. AM22₋52 exerted significant antagonistic effect on AM-induced vasorelaxation in hypoxic and normoxic conditions (apparent pK(B) = 6.8-7.2), whereas no antagonistic effect was observed in fresh and hyperoxic (95%) organ cultured vessels. The antagonistic effect exerted by αCGRP8₋37 (10⁻6·5-10⁻5·5 M) on αCGRP-induced vasodilatation in fresh vessels (derived from Schild plot pA2 = 7.4 ± 0.1) was unaltered during organ culture. The antagonistic effect exerted by αCGRP8₋37 (10⁻6 M) on AM-induced vasorelaxation in fresh vessels (apparent pK(B) = 7.4 ± 0.1) was absent during hypoxic organ culture. The receptor activity-modifying proteins 1 (RAMP1)/calcitonin-like receptor (CLR) messenger RNA ratio was reduced and RAMP2/CLR messenger RNA ratio was increased during hypoxic and normoxic organ culture compared with fresh vessels. Hypoxic organ culture for 24-72 hours potentiated the AM-induced vasorelaxation through an AM22₋52-sensitive receptor but attenuated the vasorelaxant effect of CGRP through the CGRP receptors. This could possibly be explained by relatively decreased levels of RAMP1, thus favoring RAMP2 + CLR complex (=AM receptor) formation during hypoxic organ culture.

Adrenomedullin expression in the developing human fetal lung.

BACKGROUND: Adrenomedullin (AM) is a vasodilator peptide produced by endothelial and smooth muscle cells in the systemic and pulmonary circulation. It promotes angiogenesis and alveolar growth and has protective effects in the cardiovascular and respiratory systems. Adrenomedullin's role in human pulmonary vascular and alveolar development is unknown. OBJECTIVE: To test the hypothesis that AM is expressed during normal human lung development and that its expression changes with advancing gestational age by investigating the messenger RNA and protein expression of AM and its receptor components, calcitonin-receptorlike receptor (CRLR), receptor activity-modifying protein (RAMP)2, and RAMP3 in human fetal lung from 10 to 24 weeks of gestation. METHODS: The gene expression of AM, CRLR, RAMP2, and RAMP3 was measured with real-time reverse-transcription polymerase chain reaction. Adrenomedullin protein expression was measured with Western blot. Immunohistochemical analyses of sections of lung tissue were performed. Statistical analysis was performed using linear regression and one-way analysis of variance followed by the Tukey range test. RESULTS: Adrenomedullin, CRLR, RAMP2, and RAMP3 transcripts were expressed in the midgestation human fetal lung. The gene expression of AM, CRLR, and RAMP2 increased with increasing gestational age, whereas the gene expression of RAMP3 decreased. Adrenomedullin protein expression increased with increasing gestational age. CONCLUSION: Adrenomedullin is expressed in the midgestation human fetal lung and its gene and protein expression increased with increasing gestational age, suggesting a role for AM in human lung development. Supporting this conclusion, the AM1 receptor components CRLR and RAMP2 gene expression also increased with increasing gestational age. Conversely, the expression of RAMP3, a structural component of the AM2 receptor, decreased with increasing gestational age, suggesting different functions for the AM receptors in human fetal lung, as it has been demonstrated in animal models. This speculation requires further investigation.

Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin.

Myofibroblast differentiation induced by transforming growth factor-ß (TGF-ß) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-ß) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-ß stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-ß receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-ß but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

Calcitonin receptor-like receptor expression in rat skeletal muscle fibers.

The calcitonin gene related peptide (CGRP) pathway is important in many processes including several in the central and peripheral nervous systems. CGRP is present in motor neurons and in sensory tracts, with its expression likely regulated by its use. This is supported by the fact that axotomy results in increased CGRP production in the nerve cell body. The target CGRP receptor, produced in part from the calcitonin receptor-like receptor (Calcrl) gene, has been thought to be present in multiple forms based on kinetic studies; however, understanding of the regulation of the expression of the Calcrl gene remains incomplete. CALCRL is an important factor in aging and associated disorders. This study focused on the neuromuscular system where it has been unclear whether different proteins are initially translated and whether higher levels of CALCRL are localized to the endplate regions. Rat gracilis muscle neuromuscular junctions were examined by isolating endplate enriched and non-endplate regions identified by staining for acetylcholine esterase or conjugated α-bungarotoxin binding. The CALCRL protein was detected at approximately 60kDa by Western immunoblotting and, in the isolated extracts, we found that the Calcrl mRNA level was elevated 6 fold in the muscle endplate regions and that there were two distinct Calcrl messages present. Sequence analysis showed that the two different Calcrl forms were due to alternative splicing but in a non-coding region of the transcript such that only one translation product would be generated. This indicates that previously identified pharmacologic heterogeneity is most likely due to post-translational modifications and interactions.