Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

PLCε1 suppresses tumor growth by regulating murine T cell mobilization.

Phospholipase C epsilon 1 (PLCε1) is a unique member of the phospholipase family, in that it also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1. It is this function as a Rap1 GEF that gives PLCε1 an essential role in chemokine-mediated T cell adhesion. We have utilized a syngeneic tumor model, MC38 cells in C57BL/6 mice, and observed that tumors grow larger and more quickly in the absence of PLCε1. Single-cell analysis revealed an increased CD4+ /CD8+ ratio in the spleens, lymph nodes and tumors of PLCε1 knock-out tumor-bearing mice. T cells isolated from PLCε1 knock-out mice were less activated by multiple phenotypical parameters than those from wild-type mice. We additionally noted a decrease in expression of the chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C motif chemokine receptor 4 (CCR4) on CD4+ T cells from the spleens, lymph nodes and tumors of PLCε1 knock-out mice compared to wild-type mice, and diminished migration of PLCε1-depleted CD3+ T cells towards stromal cell-derived factor (SDF)-1α. Based on these results, we conclude that PLCε1 is a potential regulator of tumor-infiltrating lymphocytes, functioning, at least in part, at the level of T cell trafficking and recruitment.

Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP.

In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

Phospholipase C zeta (PLCζ) and male infertility: Clinical update and topical developments.

The development of a mammalian embryo is initiated by a sequence of molecular events collectively referred to as 'oocyte activation' and regulated by the release of intracellular calcium in the ooplasm. Over the last decade, phospholipase C zeta (PLCζ), a sperm protein introduced into the oocyte upon gamete fusion, has gained almost universal acceptance as the protein factor responsible for initiating oocyte activation. A large body of consistent and reproducible evidence, from both biochemical and clinical settings, confers support for the role of PLCζ in this fundamental biological context, which has significant ramifications for the management of human male infertility. Oocyte activation deficiency (OAD) and total fertilisation failure (TFF) are known causes of infertility and have both been linked to abnormalities in the structure, expression, and localisation pattern of PLCζ in human sperm. Assisted oocyte activators (AOAs) represent the only therapeutic option available for OAD at present, although these agents have been the source of much debate recently, particularly with regard to their potential epigenetic effects upon the embryo. Consequently, there is much interest in the deployment of sensitive PLCζ assays as prognostic/diagnostic tests and human recombinant PLCζ protein as an alternative form of therapy. Although PLCζ deficiency has been directly linked to a cohort of infertile cases, we have yet to identify the specific causal mechanisms involved. While two genetic mutations have been identified which link defective PLCζ protein to an infertile phenotype, both were observed in the same patient, and have yet to be described in other patients. Consequently, some researchers are investigating the possibility that genetic variations in the form of single nucleotide polymorphisms (SNPs) could provide some explanation, especially since >6000 SNPs have been identified in the PLCζ gene. As yet, however, there is no consistent data to suggest that any of these SNPs influence the functional ability of PLCζ. Other laboratories appear to be focussing upon the PLCζ promoter, which is bi-directional and shared with the actin filament capping muscle Z-line alpha 3 gene (CAPZA3), or seeking to identify interacting proteins within the ooplasm. The aim of this review is to provide a synopsis of recent progress in the application of PLCζ in diagnostic and therapeutic medicine, to discuss our current understanding of how the functional ability of PLCζ might be controlled, and thus how PLCζ deficiency might arise, and finally, to consider the potential implications of alternative sperm protein candidates, such as post-acrosomal WW-domain binding protein (PAWP), which has caused much debate and confusion in the field over the last few years.

Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

PURPOSE: The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. METHODS: An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. RESULTS: Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. CONCLUSIONS: Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

PLCε knockdown inhibits prostate cancer cell proliferation via suppression of Notch signalling and nuclear translocation of the androgen receptor.

Phospholipase Cε (PLCε), a key regulator of diverse cellular functions, has been implicated in various malignancies. Indeed, PLCε functions include cell proliferation, apoptosis and malignant transformation. Here, we show that PLCε expression is elevated in prostate cancer (PCa) tissues compared to benign prostate tissues. Furthermore, PLCε depletion using an adenovirally delivered shRNA significantly decreased cell growth and colony formation, arresting the PC3 and LNCaP cell lines in the S phase of the cell cycle. We also observed that PLCε was significantly correlated with Notch1 and androgen receptor (AR). Additionally, we demonstrate that the activation of both the Notch and AR signalling pathways is involved in PLCε-mediated oncogenic effects in PCa. Our findings suggest that PLCε is a putative oncogene and prognostic marker, potentially representing a novel therapeutic target for PCa.

Phospholipase cε, an effector of ras and rap small GTPases, is required for airway inflammatory response in a mouse model of bronchial asthma.

BACKGROUND: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma. METHODS: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells. RESULTS: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency. CONCLUSIONS: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.

Knockdown of phospholipase C-epsilon by short-hairpin RNA-mediated gene silencing induces apoptosis in human bladder cancer cell lines.

Transitional cell carcinoma of bladder (TCCB) is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. To study the pathogenesis of TCCB, we investigated roles of Phospholipase C (PLC)ɛ, an effector of Ras and Rap small GTPases. RNA interference was used to knockdown PLCɛ expression in human bladder cancer cell lines (BIU-87 and T24). The expression levels of PLCɛ mRNA and protein were detected by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Flow cytometry (FCM) was used to detect distribution of cell cycle. Cellular apoptosis was reflected by transmission electron microscopy and the expression of bcl-2 and bax. We found that PLCɛ could be efficiently knocked down by shRNA. FCM assay showed that the pGenesil-PLCɛ-transfected cells were arrested at the G0/G1 phase. Silence of PLCɛ might induce apoptosis via modulation of bcl-2 and bax. In conclusion, our results suggest that PLCɛ plays an important role in the pathogenesis of human bladder cancer cells. PLCɛ may be used as a potential target of gene therapy for bladder cancer in future.

Upregulation of D2-class signaling in dopamine-denervated striatum is in part mediated by D3 receptors acting on Ca V 2.1 channels via PIP2 depletion.

The loss of dopaminergic neurons in the substantia nigra compacta followed by striatal dopamine depletion is a hallmark of Parkinson's disease. After dopamine depletion, dopaminergic D(2) receptor (D(2)R)-class supersensitivity develops in striatal neurons. The supersensitivity results in an enhanced modulation of Ca(2+) currents by D(2)R-class receptors. However, the relative contribution of D(2)R, D(3)R, and D(4)R types to the supersensitivity, as well as the mechanisms involved, have not been elucidated. In this study, whole cell voltage-clamp recordings were performed to study Ca(2+) current modulation in acutely dissociated striatal neurons obtained from rodents with unilateral 6-hydroxydopamine lesions in the substantia nigra compacta. Selective antagonists for D(2)R, D(3)R, and D(4)R types were used to identify whether the modulation by one of these receptors experiences a selective change after dopaminergic denervation. It was found that D(3)R-mediated modulation was particularly enhanced. Increased modulation targeted Ca(V)2.1 (P/Q) Ca(2+) channels via the depletion of phosphatidylinositol 4,5-bisphosphate, an intracellular signaling cascade hard to detect in control neurons and hypothesized as being amplified by dopamine depletion. An imbalance in the striatal expression of D(3)R and its splice variant, D(3)nf, accompanied enhanced D(3)R activity. Because Ca(V)2.1 Ca(2+) channels mediate synaptic GABA release from the terminals of striatal neurons, reinforcement of their inhibition by D(3)R may explain in part the profound decrease in synaptic strength in the connections among striatal projection neurons observed in the dopamine-depleted striatum.

Complete globozoospermia associated with PLCζ deficiency treated with calcium ionophore and ICSI results in pregnancy.

Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCzeta), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCzeta was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.

Crucial role of phospholipase Cepsilon in induction of local skin inflammatory reactions in the elicitation stage of allergic contact hypersensitivity.

Phospholipase Cepsilon (PLCepsilon) is an effector of Ras/Rap small GTPases. We previously demonstrated that PLCepsilon plays a crucial role in development of phorbor ester-induced skin inflammation, which is intimately involved in the promotion of skin carcinogenesis. In this study, we have examined its role in local skin inflammatory reactions during development of contact hypersensitivity toward a hapten 2,4-dinitrofluorobenzene (DNFB). PLCepsilon(+/+) and PLCepsilon(-/-) mice were sensitized with DNFB, followed by a DNFB challenge on the ears. PLCepsilon(-/-) mice exhibited substantially attenuated inflammatory reactions compared with PLCepsilon(+/+) mice as shown by suppression of ear swelling, neutrophil infiltration, and proinflammatory cytokine production. In contrast, the extent and kinetics of CD4+ T cell infiltration showed no difference depending on the PLCepsilon background. Adoptive transfer of CD4+ T cells from the sensitized mice to naive mice between PLCepsilon(+/+) and PLCepsilon(-/-) backgrounds indicated that PLCepsilon exerts its function in cells other than CD4+ T cells, presumably fibroblasts or keratinocytes of the skin, to augment inflammatory reactions during the elicitation stage of contact hypersensitivity. Moreover, dermal fibroblasts and epidermal keratinocytes cultured from the skin expressed proinflammatory cytokines in a PLCepsilon-dependent manner on stimulation with T cell-derived cytokines such as IL-17, IFN-gamma, TNF-alpha, and IL-4. These results indicate that PLCepsilon plays a crucial role in induction of proinflammatory cytokine expression in fibroblasts and keratinocytes at the challenged sites, where infiltrated CD4+ T cells produce their intrinsic cytokines, thereby augmenting the local inflammatory reactions.