Human skin keratinocytes and fibroblasts express adrenomedullin and its receptors, and adrenomedullin enhances their growth in vitro by stimulating proliferation and inhibiting apoptosis

Adrenomedullin (ADM) is a hypotensive peptide, which originates from the proteolytic cleavage of pro(p)ADM and acts via two subtypes of receptors, named L1-R (L1-R) and calcitonin-receptor-like receptor (CRLR). L1-R is selective for ADM depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of chaperones, called receptor-activity-modifying proteins (RAMPs). Compelling evidence indicates that ADM, in addition to regulating blood pressure and water and electrolyte balance, also exerts a major growth promoting action in several normal and neoplastic cells. Reverse transcription (RT)-polymerase chain reaction (PCR) allowed the detection of pADM, L1-R and CRLR mRNAs in cultured human skin keratinocytes and fibroblasts. RAMP1 and RAMP2 (but not RAMP3) mRNAs were present, but their level of expression was rather weak, thereby suggesting that L1-R is the main subtype of ADM receptor in both keratinocytes and fibroblasts. ADM concentration-dependently raised the proliferation rate and lowered the apoptotic rate of both keratinocytes and fibroblasts cultured in vitro, maximal effective concentration being 10(-8) M. The effects of 10(-8) M ADM was annulled by the putative ADM-receptor antagonist ADM22-52 (10(-6) M). ADM22-52 also lowered basal proliferative activity of keratinocytes and fibroblasts, without affecting their apoptotic deletion rate. Taken together, these findings allow us to conclude that i) human skin keratinocytes and fibroblasts express ADM and its receptors; and ii) endogenous ADM system promotes the growth of keratinocytes and fibroblasts cultured in vitro, by enhancing their proliferative activity and lowering their apoptotic deletion

Evidence for an autocrine-paracrine role of adrenomedullin in the cultured rat adrenal zona glomerulosa cells

Rat adrenomedullin (ADM) is a 50-amino acid hypotensive and vasodilating peptide, which derives from the posttranslational proteolytic cleavage of pro(p)ADM. ADM acts via at least two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as a calcitonin gene-related peptide or a selective ADM receptor depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of receptor-activity-modifying proteins (RAMPs). Adrenal zona glomerulosa (ZG) is one of the main target tissues of ADM, which has been shown to exert a potent inhibitory effect on aldosterone secretion acting through ADM[22-52]-sensitive receptors. Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pADM mRNA in the ZG, but not zona fasciculata-reticularis (ZF/R) cells of the rat adrenal cortex. Immunocytochemistry and radioimmune assay showed a weak but sizeable expression of ADM protein in the ZG, but not inner adrenocortical layers. ZG cells expressed peptidyl-glycine alpha-amidating monooxigenase, the enzyme converting immature ADM to the mature peptide, thereby suggesting their potential ability to produce active ADM. RT-PCR demonstrated the presence in ZG, but not ZF/R cells, of the specific mRNAs of L1-R, CRLR and RAMPs (especially RAMP2). ZG cells were cultured in vitro for 24 or 48 h in the presence of ADM (10(-8) M) and/or its receptor antagonist ADM[22-52] (10(-6) M). ADM increased proliferation index and lowered apoptotic index of cultured cells, and the effects were annulled by ADM[22-52]. ADM[22-52] alone was ineffective in 24 h cultures, but moderately decreased proliferation index and raised apoptotic index in 48 h cultures. In conclusion, our study provides evidence that i) rat ZG cells express ADM and ADM receptor of L1 and CRLR/RAMP2 subtypes, which both are sensitive to ADM[22-52]; and ii) endogenous ADM system modulates in an autocrine/paracrine manner ZG growth, by stimulating cell proliferation and reducing cell apoptotic deletion

Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells

Adrenomedullin (ADM) is a regulatory peptide endowed with multiple biological effects, including the regulation of blood pressure, cell growth and innate host defence. In the present study, we demonstrated that ADM signaling could be involved in the impaired cellular differentiation of myeloid leukemia cells to mature granulocytes or monocytes by modulating RAMPs/CRLR expression, PI3K/Akt cascade and the ERK/MAPK signaling pathway. When exogenously administered to in vitro cultures of HL60 promyelocytic leukemia cells, ADM was shown to exert a strong proliferative effect with minimal upregulation in the expression level of monocyte antigen CD14. Notably, the experimental inhibition of ADM signaling with inhibitor ADM22-52 promoted a differentiative stimulation towards monocytic and granulocytic lineages. Moreover, based on the expression of CD31 relative to CD38, we hypothesized that an excess of ADM in bone marrow (BM) niche could increase the transendothelial migration of leukemia cells while any inhibitory event of ADM activity could raise cell retention in hyaluronate matrix by upregulating CD38. Taken into consideration the above evidence, we concluded that ADM and ADM22-52 could differently affect the growth of leukemia cells by autocrine/paracrine mechanisms and may have clinical relevance as biological targets for the intervention of tumor progression