Adenylyl cyclase activity and gene expression during mesodermal differentiation of the P19 embryonal carcinoma cells

Abstract

DMSO-primed P19 pluripotent cells, which recapitulate the first stages of mammalian cardiogenesis and endodermal formation, were used as an in vitro model to analyze the variations in activity and expression of the different adenylyl cyclase (AC) isoforms during the early events of embryonic cell differentiation. Here, we show that the total AC activity, which increases up to 10-fold after differentiation of P19 cells, is mainly associated with increases in AC2, AC5, and AC6 mRNA levels. Particularly, the marked increase in AC5 mRNA correlates with the appearance of beating cardiomyocytes and with the transcription of the atrial myosin light chain (MLC1A) gene which encodes a protein specifically involved in the cardiac muscle cell contractile phenotype. Together, the results strongly suggest that 1) a rise in cyclic AMP (cAMP) may be associated with cardiomyocyte and endodermal cell differentiation during mammalian embryogenesis; and 2) AC5 gene expression starts very early during normal mouse cardiogenesis and correlates with the differentiation of cardiomyocytes.

Enhanced Cardiac Function in Transgenic Mice Expressing a Ca 2+ -Stimulated Adenylyl Cyclase

“…20 RNAs were hybridized with [␣- 32 P]dCTP-labeled AC8, AC5, or AC6 cDNA probes. 20 A rat GAPDH cDNA was used to control the equal RNA loading.…”

“…AC activity was measured as described 20 on purified cardiac membranes. Hearts were homogenized in 10 volumes of ice-cold lysis buffer (in mmol/L, Tris-HCl [pH 7.6] 10, EDTA 0.1, DTT 0.5, and PMSF 0.5) and centrifuged at 500g for 5 minutes at 4°C.…”

Differentiation‐induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line

“…Specifically, many studies have revealed enhancements in cAMP accumulation following increases in AC6 levels, a ubiquitously expressed isoform with integral function in the central nervous system (Lipskaia et al . 1997; Lipskaia et al . 1998; Serazin‐Leroy et al .…”

“…2002). Studies conducted in several other cell lines have demonstrated similar changes in drug‐stimulated cAMP accumulation upon differentiation that are typically accompanied by select alterations in adenylate cyclase isoform expression patterns (Lipskaia et al . 1997, 1998; Shen et al .…”

Cyclophilin A Is Required for Retinoic Acid-induced Neuronal Differentiation in p19 Cells

“…To examine whether knockdown CypA also affects Me 2 SOinduced mesodermal differentiation, we performed Western blot analysis to score the expression of GATA4 (18,19), which is expressed only in mesodermal differentiation. As shown in Fig 3E, GATA4 was expressed in Me 2 SO-treated p19 EC and S1-7 cells.…”