Expression profiles of phosphodiesterase 4D splicing variants in osteoblastic cells.
The promotion of osteoblastic differentiation by bone morphogenetic proteins (BMPs) is accelerated by chemical compounds that increase the intracellular concentration of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP is synthesized from adenosine triphosphate (ATP) by adenyl cyclase and degraded by phosphodiesterase (PDE) family enzymes. Inhibition of PDEs leads to prolonged accumulation of cAMP within cells and Camp-mediated reactions. Rolipram, a specific inhibitor of PDE4, is a compound effective in inducing osteoblastic differentiation. Four PDE4 family members are transcribed from four distinct genes (4A, 4B, 4C, and 4D). Expression of PDE4A and PDE4D has been observed in osteoblastic cells. We identified PDE4D splicing variants that expressed in ST2 or primary calvarial osteoblasts by rapid amplification of the 5'-ends of cDNA when they were cultured with BMP. PDE4D9 mRNA was identified from ST2, and PDE4D1 and -4D2 mRNAs were identified from primary calvarial osteoblasts. Expression of these three variants of PDE4D mRNA was found in ST2, MC3T3-E1, C3H10T1/2, C2C12, and primary calvarial osteoblasts by RT-PCR, but not PDE4D1 or -4D2 in ST2 or PDE4D2 in MC3T3-E1. Expression of these three variants was detectable in brain, heart, lung, liver, kidney, placenta, and femur, and was thus ubiquitous. Purified recombinant PDE4D9 protein exhibited phosphodiesterase activity, which degraded cAMP to AMP, and this activity was inhibited by rolipram. These findings suggest that PDE4D1, -2, and -9 play some roles in bone formation.
Nomura-Furuwatari
Chizumi
C
Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
Wakitani
Shigeyuki
S
Hashimoto
Yusuke
Y
Imai
Yuuki
Y
Ohta
Yoichi
Y
Nakagawa
Keisuke
K
Nakao
Yoshihiro
Y
Takayama
Kazushi
K
Manaka
Tomoya
T
Takaoka
Kunio
K
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010006:Osteoblasts / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4