Congenital Semilunar Valvulogenesis Defect in Mice Deficient in Phospholipase Cε

Abstract

Phospholipase C is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase C is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase C. These results suggest a crucial role of phospholipase C downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phosphoinositide-specific phospholipase C (PLC) is a key event triggering intracellular signal transduction from various receptor molecules at the plasma membrane by yielding two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which induce activation of protein kinase C and mobilization of Ca 2ϩ from intracellular stores, respectively (9). Concurrently, reduction in PIP 2 concentration appears to be an important signal because activities of various actin-binding proteins and pleckstrin homology domain-containing proteins are modulated through interaction with PIP 2 (25). More than 12 mammalian PLC isoforms have been identified and organized into five classes (␤, ␥, ␦, ε, and ), which are regulated through distinct mechanisms (9). PLCε is characterized by possession of two Ras-associating domains and a CDC25 homology domain. The Ras-associating domains are responsible for activation of PLCε through direct association with the GTP-bound active forms of the small GTPases Ras (15, 23), Rap1 (23,24), and Rap2 (20). Stimulation of cells by growth factors, such as platelet-derived growth factor, induces persistent activation of PLCε through activation of Ras and Rap1 (24). The rapid and initial phase of this activation is mediated by Ras at the plasma membrane, whereas Rap1 is responsible for the prolonged activation mainly at the Golgi complex (24). The CDC25 homology domain acts as a guanine nucleotide exchange factor for Rap1 and is crucial for the prolonged activation of PLCε by Rap1 (14,24). The involvement of other factors, such as the ␣ subunits of the G 12 and G 13 families or the ␤ 1 ␥ 2 subunits ...

Crucial Role of Phospholipase Cε in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

“…Mice carrying the inactivated PLCe allele (PLCe À ), created by in-frame deletion of an exon coding for the catalytic X domain, were generated as described previously (11,14). They were backcrossed to C57BL/6 strain eight times.…”

“…Reverse transcription-PCR (RT-PCR) was performed as described previously (11,24). The sequences of the primers used for PCR are listed in Supplementary Table S1.…”

“…Ear thickness was measured with calipers, and ear swelling was calculated as (thickness at each time point) À (thickness at 0 h). Histologic analysis with H&E staining and immunohistochemical analysis of the skin sections were carried out essentially as described (11,14).…”

“…Knockdown of the PLCe orthologue in the zebrafish resulted in loss of the filtration barrier in the glomerular podocytes resembling the human symptom (10). Mice homozygous for the functionally inactivated PLCe allele (PLCe À/À mice) exhibited semilunar valvulogenesis defect, leading to cardiac dilation (11), and mice with total disruption of the PLCe gene developed cardiac hypertrophy under an extreme cardiac stress (12). Targeted inactivation of the PLCe orthologue in the nematode Caenorhabditis elegans resulted in delayed dilation of the spermatheca-uterine valve, leading to defective ovulation (13).…”

Phospholipase Cɛ has a crucial role in ultraviolet B-induced neutrophil-associated skin inflammation by regulating the expression of CXCL1/KC

“…17 Pathophysiological roles of PLCe have been studied in various animal models carrying artificial or spontaneous mutations in the chromosomal gene. [23][24][25] Also, using positional cloning, the human PLCe gene PLCE1 was identified as a causal gene for nephrotic syndrome. 26 However, few studies on the role of PLCe in external stimuliinduced pathophysiological conditions have been carried out.…”