Ginsenoside Re Protects against Serotonergic Behaviors Evoked by 2,5-Dimethoxy-4-iodo-amphetamine in Mice via Inhibition of PKCδ-Mediated Mitochondrial Dysfunction.

It has been recognized that serotonin 2A receptor (5-HT2A) agonist 2,5-dimethoxy-4-iodo-amphetamine (DOI) impairs serotonergic homeostasis. However, the mechanism of DOI-induced serotonergic behaviors remains to be explored. Moreover, little is known about therapeutic interventions against serotonin syndrome, although evidence suggests that ginseng might possess modulating effects on the serotonin system. As ginsenoside Re (GRe) is well-known as a novel antioxidant in the nervous system, we investigated whether GRe modulates 5-HT2A receptor agonist DOI-induced serotonin impairments. We proposed that protein kinase Cδ (PKCδ) mediates serotonergic impairments. Treatment with GRe or 5-HT2A receptor antagonist MDL11939 significantly attenuated DOI-induced serotonergic behaviors (i.e., overall serotonergic syndrome behaviors, head twitch response, hyperthermia) by inhibiting mitochondrial translocation of PKCδ, reducing mitochondrial glutathione peroxidase activity, mitochondrial dysfunction, and mitochondrial oxidative stress in wild-type mice. These attenuations were in line with those observed upon PKCδ inhibition (i.e., pharmacologic inhibitor rottlerin or PKCδ knockout mice). Furthermore, GRe was not further implicated in attenuation mediated by PKCδ knockout in mice. Our results suggest that PKCδ is a therapeutic target for GRe against serotonergic behaviors induced by DOI.

Impaired respiratory burst contributes to infections in PKCδ-deficient patients.

Patients with autosomal recessive protein kinase C δ (PKCδ) deficiency suffer from childhood-onset autoimmunity, including systemic lupus erythematosus. They also suffer from recurrent infections that overlap with those seen in patients with chronic granulomatous disease (CGD), a disease caused by defects of the phagocyte NADPH oxidase and a lack of reactive oxygen species (ROS) production. We studied an international cohort of 17 PKCδ-deficient patients and found that their EBV-B cells and monocyte-derived phagocytes produced only small amounts of ROS and did not phosphorylate p40phox normally after PMA or opsonized Staphylococcus aureus stimulation. Moreover, the patients' circulating phagocytes displayed abnormally low levels of ROS production and markedly reduced neutrophil extracellular trap formation, altogether suggesting a role for PKCδ in activation of the NADPH oxidase complex. Our findings thus show that patients with PKCδ deficiency have impaired NADPH oxidase activity in various myeloid subsets, which may contribute to their CGD-like infectious phenotype.

PKCδ deficiency inhibits fetal development and is associated with heart elastic fiber hyperplasia and lung inflammation in adult PKCδ knockout mice.

Protein kinase C-delta (PKCδ) has a caspase-3 recognition sequence in its structure, suggesting its involvement in apoptosis. In addition, PKCδ was recently reported to function as an anti-cancer factor. The generation of a PKCδ knockout mouse model indicated that PKCδ plays a role in B cell homeostasis. However, the Pkcrd gene, which is regulated through complex transcription, produces multiple proteins via alternative splicing. Since gene mutations can result in the loss of function of molecular species required for each tissue, in the present study, conditional PKCδ knockout mice lacking PKCδI, II, IV, V, VI, and VII were generated to enable tissue-specific deletion of PKCδ using a suitable Cre mouse. We generated PKCδ-null mice that lacked whole-body expression of PKCδ. PKCδ+/- parental mice gave birth to only 3.4% PKCδ-/- offsprings that deviated significantly from the expected Mendelian ratio (χ2(2) = 101.7, P < 0.001). Examination of mice on embryonic day 11.5 (E11.5) showed the proportion of PKCδ-/- mice implanted in the uterus in accordance with Mendelian rules; however, approximately 70% of the fetuses did not survive at E11.5. PKCδ-/- mice that survived until adulthood showed enlarged spleens, with some having cardiac and pulmonary abnormalities. Our findings suggest that the lack of PKCδ may have harmful effects on fetal development, and heart and lung functions after birth. Furthermore, our study provides a reference for future studies on PKCδ deficient mice that would elucidate the effects of the multiple protein variants in mice and decipher the roles of PKCδ in various diseases.

Endothelial deletion of PKCδ prevents VEGF inhibition and restores blood flow reperfusion in diabetic ischemic limb.

AIMS: Peripheral artery disease is a complication of diabetes leading to critical hindlimb ischemia. Diabetes-induced inhibition of VEGF actions is associated with the activation of protein kinase Cδ (PKCδ). We aim to specifically investigate the role of PKCδ in endothelial cell (EC) function and VEGF signaling. METHODS: Nondiabetic and diabetic mice, with (ec-Prkcd-/-) or without (ec-Prkcdf/f) endothelial deletion of PKCδ, underwent femoral artery ligation. Blood flow reperfusion was assessed up to 4 weeks post-surgery. Capillary density, EC apoptosis and VEGF signaling were evaluated in the ischemic muscle. Src homology region 2 domain-containing phosphatase-1 (SHP-1) phosphatase activity was assessed in vitro using primary ECs. RESULTS: Ischemic muscle of diabetic ec-Prkcdf/f mice exhibited reduced blood flow reperfusion and capillary density while apoptosis increased as compared to nondiabetic ec-Prkcdf/f mice. In contrast, blood flow reperfusion and capillary density were significantly improved in diabetic ec-Prkcd-/- mice. VEGF signaling pathway was restored in diabetic ec-Prkcd-/- mice. The deletion of PKCδ in ECs prevented diabetes-induced VEGF unresponsiveness through a reduction of SHP-1 phosphatase activity. CONCLUSIONS: Our data provide new highlights in mechanisms by which PKCδ activation in EC contributed to poor collateral vessel formation, thus, offering novel therapeutic targets to improve angiogenesis in the diabetic limb.

PKC-δ deficiency in B cells displays osteopenia accompanied with upregulation of RANKL expression and osteoclast-osteoblast uncoupling.

PKC-δ is an important molecule for B-cell proliferation and tolerance. B cells have long been recognized to play a part in osteoimmunology and pathological bone loss. However, the role of B cells with PKC-δ deficiency in bone homeostasis and the underlying mechanisms are unknown. We generated mice with PKC-δ deletion selectively in B cells by crossing PKC-δ-loxP mice with CD19-Cre mice. We studied their bone phenotype using micro-CT and histology. Next, immune organs were obtained and analyzed. Western blotting was used to determine the RANKL/OPG ratio in vitro in B-cell cultures, ELISA assay and immunohistochemistry were used to analyze in vivo RANKL/OPG balance in serum and bone sections respectively. Finally, we utilized osteoclastogenesis to study osteoclast function via hydroxyapatite resorption assay, and isolated primary calvaria osteoblasts to investigate osteoblast proliferation and differentiation. We also investigated osteoclast and osteoblast biology in co-culture with B-cell supernatants. We found that mice with PKC-δ deficiency in B cells displayed an osteopenia phenotype in the trabecular and cortical compartment of long bones. In addition, PKC-δ deletion resulted in changes of trabecular bone structure in association with activation of osteoclast bone resorption and decrease in osteoblast parameters. As expected, inactivation of PKC-δ in B cells resulted in changes in spleen B-cell number, function, and distribution. Consistently, the RANKL/OPG ratio was elevated remarkably in B-cell culture, in the serum and in bone specimens after loss of PKC-δ in B cells. Finally, in vitro analysis revealed that PKC-δ ablation suppressed osteoclast differentiation and function but co-culture with B-cell supernatant reversed the suppression effect, as well as impaired osteoblast proliferation and function, indicative of osteoclast-osteoblast uncoupling. In conclusion, PKC-δ plays an important role in the interplay between B cells in the immune system and bone cells in the pathogenesis of bone lytic diseases.

Protein kinase Cδ knockout mice are protected from cocaine-induced hepatotoxicity.

We investigated whether protein kinase Cδ (PKCδ) mediates cocaine-induced hepatotoxicity in mice. Cocaine treatment (60 mg/kg, i.p.) significantly increased cleaved PKCδ expression in the liver of wild-type (WT) mice, and led to significant increases in oxidative parameters (i.e., reactive oxygen species, 4-hydroxylnonenal and protein carbonyl). These cocaine-induced oxidative burdens were attenuated by pharmacological (i.e., rottlerin) or genetic depletion of PKCδ. We also demonstrated that treatment with cocaine resulted in significant increases in nuclear factor erythroid-2-related factor 2 (Nrf-2) nuclear translocation and increased Nrf-2 DNA-binding activity in wild-type (WT) mice. These increases were more pronounced in the rottlerin-treated WT or PKCδ knockout mice than in the saline-treated WT mice. Although cocaine treatment increased Nrf-2 nuclear translocation, DNA binding activity, and γ-glutamyl cysteine ligases (i.e., GCLc and GCLm) mRNA expressions, while it reduced the glutathione level and GSH/GSSG ratio. These decreases were attenuated by PKCδ depletion. Cocaine treatment significantly increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of WT mice signifying the hepatic damage. These increases were also attenuated by PKCδ depletion. In addition, cocaine-induced hepatic degeneration in WT mice was evident 1 d post-cocaine. At that time, cocaine treatment decreased Bcl-2 and Bcl-xL levels, and increased Bax, cytosolic cytochrome c, and cleaved caspase-3 levels. Pharmacological or genetic depletion of PKCδ significantly ameliorated the pro-apoptotic properties and hepatic degeneration. Therefore, our results suggest that inhibition of PKCδ, as well as activation of Nrf-2, is important for protecting against hepatotoxicity induced by cocaine.

α-Ketoglutarate stimulates pendrin-dependent Cl- absorption in the mouse CCD through protein kinase C.

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a Gq protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl-/[Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl- absorption, we examined α-KG-stimulated Cl- absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl- absorption in CCDs from wild-type mice but had no effect on Cl- absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl- absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl- absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl- absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca2+ chelators (BAPTA, 10-20 µM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl- absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl- absorption. However, in SPAK null CCDs, Cl- absorption was not activated further by luminal α-KG application nor was Cl- absorption reduced with the PKC inhibitor GF109203 . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl-/[Formula: see text] exchange through a mechanism dependent on PKC and Ca2+ that involves PKC-α and PKC-δ.

Multisite phosphorylation of P-Rex1 by protein kinase C.

P-Rex proteins are guanine nucleotide exchange factors (GEFs) that act on the Rho/Rac family of GTP binding proteins. The activity of P-Rex proteins is regulated by several extracellular stimuli. In fact, activation of growth factor receptors has been reported to activate a phosphorylation/dephosphorylation cycle of P-Rex1. Such cycle includes dephosphorylation of serines 313 and 319 which negatively regulate the GEF activity of P-Rex1, together with phosphorylation of serines 605 and 1169 which favour P-Rex1 GEF activity. However, the kinases that regulate phosphorylation at these different regulatory sites are largely unknown. Here we have investigated the potential regulatory action of several kinases on the phosphorylation of P-Rex1 at S313, S319, S605 and S1169. We show that activation of protein kinase C (PKC) caused phosphorylation of S313, S319 and S1169. Activation of growth factor receptors induced phosphorylation of S1169 through a mechanism that was independent of PKC, indicating that distinct kinases and mechanisms control the phosphorylation of P-Rex1 at different regulatory serines. Genetic and biochemical studies confirmed that the PKC isoform PKCδ was able to directly phosphorylate P-Rex1 at S313. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S313 was substituted by alanine, indicated that phosphorylation at that residue negatively regulated P-Rex1 exchange activity. We suggest that control of P-Rex1 activity depends on a highly dynamic interplay among distinct signalling routes and its multisite phosphorylation is controlled by the action of different kinases.

PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice.

Para-methoxymethamphetamine (PMMA) is a para-ring-substituted amphetamine derivative sold worldwide as an illegal psychotropic drug. Although PMMA use has been reported to lead to severe intoxication and even death, little is known about the mechanism(s) by which PMMA exerts its neurotoxic effects. Here we found that PMMA treatment resulted in phosphorylation of protein kinase Cδ (PKCδ) and subsequent mitochondrial translocation of cleaved PKCδ. PMMA-induced oxidative stress was more pronounced in mitochondria than in the cytosol. Moreover, treatment with PMMA consistently resulted in significant reductions in mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial Mn superoxide dismutase-immunoreactivity. In contrast, PMMA treatment led to a significant increase in intramitochondrial Ca2+ level. Treatment with PMMA also significantly increased ionized calcium binding adaptor molecule 1 (Iba-1)-labeled microglial activation and upregulated tumor necrosis factor alpha (TNF-α) gene expression. PKCδ knockout attenuated these mitochondrial effects and dampened the neurotoxic effects of PMMA. Importantly, TNF-α knockout mice were significantly protected from PMMA-induced increases in phospho-PKCδ expression, mitochondrial translocation of cleaved PKCδ, and Iba-1-labeled microgliosis. Both rottlerin, a pharmacological inhibitor of PKCδ, and etanercept, a pharmacological inhibitor of TNF-α, significantly protected against PMMA-mediated induction of apoptosis, as assessed by terminal deoxynucleotidyl transferase dUDP nick end labeling (TUNEL) assays. In addition, PKCδ knockout and TNF-α knockout both resulted in decreased PMMA-mediated induction of dopaminergic loss. Therefore, our results suggest that PKCδ mediates PMMA-induced neurotoxicity by facilitating oxidative stress (mitochondria > cytosol), mitochondrial dysfunction, microglial activation, and pro-apoptotic signaling. Our results also indicate that PMMA-induced PKCδ activation requires the proinflammatory cytokine TNF-α.

Loss of PKCδ Induces Prostate Cancer Resistance to Paclitaxel through Activation of Wnt/ß-Catenin Pathway and Mcl-1 Accumulation.

Prostate cancer is the leading cause of cancer-related death among men in developed countries. Although castration therapy is initially effective, prostate cancers progress to hormone-refractory disease and in this case taxane-based chemotherapy is widely used. Castration-resistant prostate cancer cells often develop resistance to chemotherapy agents and the search for new therapeutic strategies is necessary. In this article, we demonstrate that PKCδ silencing favors mitotic arrest after paclitaxel treatment in PC3 and LNCaP cells; however, this is associated with resistance to paclitaxel-induced apoptosis. In prostate cancer cells, PKCδ seems to exert a proapoptotic role, acting as a negative regulator of the canonical Wnt/ß-catenin pathway. PKCδ silencing induces activation of Wnt/ß-catenin pathway and the expression of its target genes, including Aurora kinase A, which is involved in activation of Akt and both factors play a key role in GSK3ß inactivation and consequently in the stabilization of ß-catenin and antiapoptotic protein Mcl-1. We also show that combined treatments with paclitaxel and Wnt/ß-catenin or Akt inhibitors improve the apoptotic response to paclitaxel, even in the absence of PKCδ. Finally, we observe that high Gleason score prostate tumors lose PKCδ expression and this correlates with higher activation of ß-catenin, inactivation of GSK3ß, and higher levels of Aurora kinase A and Mcl-1 proteins. These findings suggest that targeting Wnt/ß-catenin or Akt pathways may increase the efficacy of taxane chemotherapy in advanced human prostate cancers that have lost PKCδ expression. Mol Cancer Ther; 15(7); 1713-25. ©2016 AACR.

Lack of protein kinase C-delta (PKCδ) disrupts fertilization and embryonic development.

This study tested the function of protein kinase C delta (PKCδ) during fertilization and embryonic development using gene-knockout (Prkcd(-/-)) mice. Fertility analysis revealed that Prkcd(-/-) mating pairs produce significantly fewer pups per litter than wild-type pairs (P < 0.05), and exhibit a high incidence of embryonic loss post-implantation. Both Prkcd(-/-) male as well as Prkcd(-/-) female mice mated to Prkcd(+/+) controls also showed reduced litter sizes, with a selective loss of Prkcd-null pups. Further analysis of the females demonstrated comparable in vitro fertilization outcomes between control and Prkcd(-/-) oocytes fertilized with wild-type sperm. Pregnant Prkcd(-/-) females, however, exhibited a reduced number of total implantations, suggesting a possible disruption in early embryo quality and/or implantation. In turn, male gamete analysis revealed that Prkcd(-/-) sperm demonstrated a decreased capacity to penetrate the zona pellucida (P < 0.05), necessary for successful fertilization. Moreover, we identified phosphorylated PKCδ as a component of the sperm acrosome, indicating a potential role for this kinase in acrosome exocytosis. Therefore, loss of PKCδ disrupts key reproductive functions in both males and females that limit fertility.

Protein kinase C δ deficiency enhances megakaryopoiesis and recovery from thrombocytopenia.

OBJECTIVE: We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. APPROACH AND RESULTS: Using PKCδ(-/-) and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes (P<0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ(-/-) mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type (P<0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ(-/-) megakaryocytes compared with wild-type. Finally, PKCδ(-/-) mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. CONCLUSIONS: These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.

Protein kinase Cδ promotes transitional B cell-negative selection and limits proximal B cell receptor signaling to enforce tolerance.

Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca(2+)-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation.

Inhibition of protein kinase C delta attenuates allergic airway inflammation through suppression of PI3K/Akt/mTOR/HIF-1 alpha/VEGF pathway.

Vascular endothelial growth factor (VEGF) is supposed to contribute to the pathogenesis of allergic airway disease. VEGF expression is regulated by a variety of stimuli such as nitric oxide, growth factors, and hypoxia-inducible factor-1 alpha (HIF-1α). Recently, inhibition of the mammalian target of rapamycin (mTOR) has been shown to alleviate cardinal asthmatic features, including airway hyperresponsiveness, eosinophilic inflammation, and increased vascular permeability in asthma models. Based on these observations, we have investigated whether mTOR is associated with HIF-1α-mediated VEGF expression in allergic asthma. In studies with the mTOR inhibitor rapamycin, we have elucidated the stimulatory role of a mTOR-HIF-1α-VEGF axis in allergic response. Next, the mechanisms by which mTOR is activated to modulate this response have been evaluated. mTOR is known to be regulated by phosphoinositide 3-kinase (PI3K)/Akt or protein kinase C-delta (PKC δ) in various cell types. Consistent with these, our results have revealed that suppression of PKC δ by rottlerin leads to the inhibition of PI3K/Akt activity and the subsequent blockade of a mTOR-HIF-1α-VEGF module, thereby attenuating typical asthmatic attack in a murine model. Thus, the present data indicate that PKC δ is necessary for the modulation of the PI3K/Akt/mTOR signaling cascade, resulting in a tight regulation of HIF-1α activity and VEGF expression. In conclusion, PKC δ may represent a valuable target for innovative therapeutic treatment of allergic airway disease.

Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing.

Potassium conductance in vascular smooth muscle (VSM) is known to be altered in arterial hypertension. High level of protein kinase C (PKC) activity is a common feature for hypertension of different genesis. The main goal of this study was to investigate the efficacy of the RNA interference (RNAi) technique targeting PKC delta-isoform gene as a possible pharmacological tool to restore vasodilator potential in spontaneously hypertensive rats (SHR). Experimental design of the study comprised RNAi and patch-clamp techniques, RT-PCR analysis and standard acetylcholine test. Total outward currents and acetylcholine-induced endothelium-dependent relaxant responses were blunted in SHR. BKCa alpha subunit mRNA expression in SHR was unchanged whereas KV and KATP mRNA expression appeared significantly increased. PKC inhibitor, chelerythrine (100 nM), restored potassium channels activity in SHR. PKC-delta-isoform protein expression and PKC-delta-isoform mRNA expression are 2.5-4 fold increased in VSM from SHR. PKC gene silencing with the short hairpin RNAs (shRNAs)-plasmid delivery system administered intravenously led to an increment in maximal amplitude of acetylcholine-relaxation, restored outward K(+) currents and PKC-delta-isoform mRNA and protein expression. Arterial blood pressure in SHR was normalized following shRNAs administration. We conclude that BKCa channels are likely to be the most PKC-dependent member of K(+) channels family responsible for vascular hypercontractility in SHR while Kv and KATP channels may constitute a reserve mechanism for the maintenance of vasodilator potential under BKCa channelopathy. It is likely that RNAi technique is a good therapeutic approach to inactivate PKC gene and to normalize vascular functions and high arterial blood pressure in SHR.

Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS: We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS: We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.