Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

Abstract

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the ␣ subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5′-AGGCTAC-CACAGTCTCCCTG-3′ and 5′-GCAGTGTAAAATCTCCAGTC-3′ (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5′-CTGCCTTTATG GATGATGGT-3′ (sense) and 5′-GTTCAGCTCGAAGAAGA-3′ (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells. Leukemia (2000) 14, 1097-1104.

Mineralocorticoid Receptor-Mediated Signaling Regulates the Ion Gated Sodium Channel in Vascular Endothelial Cells and Requires an Intact Cytoskeleton

“…1 show that the PCR products were finally resolved as predicted bands of 843 and 520 bp for the MCR (lane b) and the ENaC BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (lane c), respectively, on agarose gels as in previous studies with cells and tissues known to respond to the MCR (4, 5, 8 -12). The amplification by a primer similar to that used in earlier studies (8,12,14) suggests that the initiation sites for the MCR and ENaC are closely related in cells of diverse embryological origins. Sequence analysis of the residues 1-778 in the PCR products revealed almost 100% homology with the residues between 2944 (5Ј end) and 2525 (3Ј end) of human kidney MCR gene with only minor substitutions (data not shown).…”

“…FITC was excited at 488 nm by a 20 mW air cooled argon laser through a dichroic miror and the emmission was recorded in green after passage through a 505 nm/550 nm band pass filter. The FITC and TRITC images were recorded alternatively in the same field and then superimposed in yellow to assess the colocalization of the MCR and ENaC with actin or tubulin (12).…”

“…Cell RNA was prepared according to the protocol of Trizol Reagent (Gibco BRL) which is an improvement over the single step RNA isolation method (8,12,13). Briefly, the cells (5 ϫ 10 6 ) were lysed with 1 ml of Trizol and the lysate was passed several times through a pipette.…”

“…RNA was denatured for 10 min at 70°C and incubated for 60 min at 42°C in a final volume of 20 l in the presence of 0.5 mM dNTP (Pharmacia), 0.01 M dithiothreitol, 10 pM oligo(dT) (Promega), 200 units Superscript II RNAse H Ϫ reverse transcriptase (BRL), and 1 unit RNAsin (Promega) in 50 mM Tris-HCl, pH 8.3, containing 75 mM KCl and 3 mM MgCl 2 . The mixture was heated to 95°C for 5 min at the end of the reaction, as described in our recent studies (8,12,13). The polymerase chain reaction (PCR) for the MCR was performed with the aid of the primers 5Ј-AGGCTACCACAGTCTCCCTG-3Ј (sense) and 5Ј-GCAGTG TAAAATCTCCAGTC-3Ј (antisense) synthesized by Genosys (U.K.).…”

“…The PCR products (10 l) were electrophoresed on 1.2% agarose gels in 0.45 mM Tris-borate containing 0.2% ethidium bromide and 1 mM EDTA. A negative H 2 O control was similarly amplified in parallel to check the efficiency of the PCR technique (8,12,13). The gels were photographed along with the molecular weight markers run concurrently.…”