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Five million non-melanoma skin cancers occur globally each year, and it is one of the most common malignant cancers. The dysregulation of the endocannabinoid system, particularly cannabinoid receptor 2 (CB2), is implicated in skin cancer development, progression, and metastasis. Comparing wildtype (WT) to systemic CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in one-year old mice, and subsequently used the multi-stage chemical carcinogenesis model, wherein cancer is initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that aging CB2-/- mice have an increased incidence of spontaneous cancerous and precancerous skin lesions compared to their WT counterparts. In the DMBA/TPA model, CB2-/- developed more and larger papillomas, had decreased spontaneous regression of papillomas, and displayed an altered systemic immune profile, including upregulated CD4+ T cells and dendritic cells, compared to WT mice. Immune cell infiltration in the tumor microenvironment was generally low for both genotypes, although a trend of higher myeloid-derived suppressor cells was observed in the CB2-/- mice. CB2 expression in carcinogen-exposed skin was significantly higher compared to naïve skin in WT mice, suggesting a role of CB2 on keratinocytes. Taken together, our data show that endogenous CB2 activation plays an anti-tumorigenic role in non-melanoma skin carcinogenesis, potentially via an immune-mediated response involving the alteration of T cells and myeloid cells coupled with the modulation of keratinocyte activity.

The endocannabinoid system, particularly cannabinoid receptor 2 (CB2 in mice and CNR2 in humans), has controversial pathophysiological implications in colon cancer. Here, we investigate the role of CB2 in potentiating the immune response in colon cancer in mice and determine the influence of CNR2 variants in humans. Comparing wild-type (WT) mice to CB2 knockout (CB2−/−) mice, we performed a spontaneous cancer study in aging mice and subsequently used the AOM/DSS model of colitis-associated colorectal cancer and a model for hereditary colon cancer (ApcMin/+). Additionally, we analyzed genomic data in a large human population to determine the relationship between CNR2 variants and colon cancer incidence. Aging CB2−/− mice exhibited a higher incidence of spontaneous precancerous lesions in the colon compared to WT controls. The AOM/DSS-treated CB2−/− and ApcMin/+CB2−/− mice experienced aggravated tumorigenesis and enhanced splenic populations of immunosuppressive myeloid-derived suppressor cells along with abated anti-tumor CD8+ T cells. Importantly, corroborative genomic data reveal a significant association between non-synonymous variants of CNR2 and the incidence of colon cancer in humans. Taken together, the results suggest that endogenous CB2 activation suppresses colon tumorigenesis by shifting the balance towards anti-tumor immune cells in mice and thus portray the prognostic value of CNR2 variants for colon cancer patients.

Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1Cre EPORf/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysMCre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115+) observed following high-dose EPO administration and the resulting bone loss in LysMCre EPORf/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass.

Erythropoietin (EPO) is a key regulator of erythropoiesis. However, EPO receptors (EPO-Rs) are also expressed on non-erythroid cell types, including myeloid and bone cells. Immune cells also participate in bone homeostasis. B cells produce receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG), two pivotal regulators of bone metabolism. Here we explored the ability of B cells to transdifferentiate into functional osteoclasts and examined the role of EPO in this process in a murine model. Methods: We have combined specifically-designed experimental mouse models and in vitro based osteoclastogenesis assays, as well as PCR analysis of gene expression. Results: (i) EPO treatment in vivo increased RANKL expression in bone marrow (BM) B cells, suggesting a paracrine effect on osteoclastogenesis; (ii) B cell-derived osteoclastogenesis occured in vivo and in vitro, as demonstrated by B cell lineage tracing in murine models; (iii) B-cell-derived osteoclastogenesis in vitro was restricted to Pro-B cells expressing CD115/CSF1-R and is enhanced by EPO; (iv) EPO treatment increased the number of B-cell-derived preosteoclasts (β3+CD115+), suggesting a physiological rationale for B cell derived osteoclastogenesis; (v) finally, mice with conditional EPO-R knockdown in the B cell lineage (cKD) displayed a higher cortical and trabecular bone mass. Moreover, cKD displayed attenuated EPO-driven trabecular bone loss, an effect that was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling and suggests their involvement in the regulation of bone homeostasis and possibly in EPO-stimulated erythropoietic response. Importantly, we present here for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo.

The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin−CD11b−Ly6Chi CD115+, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment.

Krox20/EGR2 is a zinc finger transcription factor, implicated in the development of the hindbrain, nerve myelination, and tumor suppression. In skeletal biology, we have demonstrated that Krox20 also regulates adult bone metabolism. We and others have characterized several functions of Krox20 in the osteoclast lineage, namely, preosteoclast proliferation and differentiation, and mature osteoclast apoptosis. We have previously reported that systemically Krox20-haploinsufficient mice have a low bone mass with increased bone resorption. However, new data have now revealed that this phenotype is restricted to females. In addition, we discovered that conditional knockout of Krox20 (cKO) restricted to osteoclast progenitors is sufficient to induce the same female-specific bone loss observed in systemic mutants. To test whether this sexual dimorphism results from an interaction between Krox20 and sex hormones, we examined the sex- and hormone-dependent role of Krox20 deficiency on proliferation and apoptosis in osteoclastic cells. Our results indicate that male and female sex hormones (dihydrotestosterone [DHT] and estradiol [E2], respectively) as well as Krox20 inhibit preosteoclast proliferation and augment osteoclast apoptosis. The observation that Krox20 expression is inhibited by DHT and E2 negates the hypothesis that the effect of sex hormones is mediated by an increase in Krox20 expression. Interestingly, the effect of Krox20 deficiency was observed only with cells derived from female animals, regardless of any sex hormones added in vitro. In addition, we have identified sexual dimorphism in the expression of several Krox20-related genes, including NAB2. This sex-specific epigenetic profile was established at puberty, maintained in the absence of sex hormones, and explains the female-specific skeletal importance of Krox20. The findings described in this study emphasize the medical importance of sex differences, which may be determined at the epigenetic level. © 2019 American Society for Bone and Mineral Research.

Recent studies have demonstrated that erythropoietin (EPO) treatment in mice results in trabecular bone loss. Here, we investigated the dose-response relationship between EPO, hemoglobin (Hgb) and bone loss and examined the reversibility of EPO-induced damage. Increasing doses of EPO over two weeks led to a dose-dependent increase in Hgb in young female mice, accompanied by a disproportionate decrease in trabecular bone mass measured by micro-CT (µCT). Namely, increasing EPO from 24 to 540 IU/week produced a modest 12% rise in Hgb (20.2 ± 1.3 mg/dL vs 22.7 ± 1.3 mg/dL), while trabecular bone volume fraction (BV/TV) in the distal femur decreased dramatically (27 ± 8.5% vs 53 ± 10.2% bone loss). To explore the long-term skeletal effects of EPO, we treated mice for two weeks (540 IU/week) and monitored bone mass changes after treatment cessation. Six weeks post-treatment, there was only a partial recovery of the trabecular microarchitecture in the femur and vertebra. EPO-induced bone loss is therefore dose-dependent and mostly irreversible at doses that offer only a minor advantage in the treatment of anemia. Because patients requiring EPO therapy are often prone to osteoporosis, our data advocate for using the lowest effective EPO dose for the shortest period of time to decrease thromboembolic complications and minimize the adverse skeletal outcome.

Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro. Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A). To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups. In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B). Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO. This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The inbred mouse strain C57BL/6 is commonly used for the generation of transgenic mouse and is a well established strain in bone research. Different vendors supply different substrains of C57BL/6J as wild-type animals when genetic drift did not incur any noticeable phenotype. However, we sporadically observed drastic differences in the bone phenotype of "WT" C57BL/6J mice originating from different labs and speculated that these variations are attributable, at least in part, to the variation between C57BL/6J substrains, which is often overlooked. C57BL/6J-OlaHsd is a commonly used substrain that despite a well defined deletion in the alpha-synuclein (Snca) and multimerin-1 (Mmrn1) genes, was reported to display no obvious phenotype and is used as WT control. Here, we compared the bone phenotype of C57BL/6J-OlaHsd (6J-OLA) to C57BL/6J-RccHsd (6J-RCC) and to the original C57BL/6J (6J-JAX). Using μCT analysis, we found that 6J-OLA mice display a significantly lower trabecular bone mass compared to 6J-RCC and 6J-JAX. PCR analysis revealed that both the Snca and Mmrn1 genes are expressed in bone tissue of 6J-RCC animals but not of 6J-OLA mutants, suggesting either one or both genes play a role in bone metabolism. In vitro analysis demonstrated increase in osteoclasts number and decreased osteoblast mineralization in cells derived from 6J-OLA compared with 6J-RCC. Our data may shed light on unexplained differences in basal bone measurements between different research centers and reiterate the importance of specifying the exact substrain type. In addition, our findings describe the physiological role for Mmrn1 and/or Snca in bone remodeling.

During development, enhancers play pivotal roles in regulating gene expression programs; however, their involvement in cancer progression has not been fully characterized. We performed an integrative analysis of DNA methylation, RNA-seq, and small RNA-seq profiles from thousands of patients, including 25 diverse primary malignances and seven body sites of metastatic melanoma. We found that enhancers are consistently the most differentially methylated regions (DMR) as cancer progresses from normal to primary tumors and then to metastases, compared to other genomic features. Remarkably, identification of enhancer DMRs (eDMRs) enabled classification of primary tumors according to physiological organ systems, and in metastasis eDMRs are the most correlated with patient outcome. To further understand the eDMR role in cancer progression, we developed a model to predict genes and microRNAs that are regulated by enhancer and not promotor methylation, which shows high accuracy with chromatin architecture methods and was experimentally validated. Interestingly, among all metastatic melanoma eDMRs, the most correlated with patient survival were eDMRs that “switched” their methylation patterns back and forth between normal, primary, and metastases and target cancer drivers, e.g., KIT. We further demonstrated that eDMR target genes were modulated in melanoma by the bone metastasis microenvironment, suggesting that eDMRs respond to microenvironmental cues in metastatic niches. Our findings that aberrant methylation in cancer cells mostly affects enhancers, which contribute to tumor progression and cancer cell plasticity, will facilitate development of epigenetic anticancer approaches.

Background and aims: Immunotherapy with anti-CD20-specific antibodies (e.g. rituximab), has become the standard of care for B cell lymphoproliferative disorders and many autoimmune diseases. Despite previously demonstrated role for B cells in bone metabolism, the effect of anti-CD20-mediated B cell depletion on bone mass in human patients has not been thoroughly studied. For example, in rheumatological patients the effect of rituximab on bone mass yielded conflicting results, while in lymphoma patients it has not yet been described. Here, we describe the effect of treatment with anti-CD20-specific antibodies on bone mass in a cohort of patients with follicular lymphoma and propose a plausible mechanism using murine model. Methods: To assess the effect of rituximab on bone mass in lymphoma patients, we retrospectively studied the bone mass in patients with follicular lymphoma (FL) during the maintenance phase of chemoimunotherapy, i.e. when rituximab is administered as monotherapy. FL patients on no maintenance (historical controls) or patients with marginal zone lymphoma were include as a control group. Cross-sectional X-ray imaging (CT/PET-CT), performed at the completion of the induction phase and 6-12 months thereafter, were used to serially assess bone density. Wild-type female C57BL/6J mice and mouse anti-mouse CD20 antibody (Genentech) were used for the animal experimental system. Murine bone structure was assessed by the microCT method. Immunophenotyping of the bone marrow (BM), spleen and peripheral blood cells was performed. ELISA and "real-time" quantitative PCR were used to measure the levels of key mediators of bone remodeling, e.g. RANKL, OPG and TNFα. Standard osteoclastogenic assay was used to assesses the osteoclastogenic potential of BM cells. Results: Rituximab treatment prevented the decline in bone mass observed in patients who did not receive active maintenance therapy, both in the lumbar spine (-2.6% vs -8%) and femoral head (-0.5% vs -5.1%) (n=12 patients in each group, p Conclusions: While many lymphoma patients may suffer from bone loss due to advanced age and glucocorticoid administration, our data suggest additional favorable effect of anti-CD20 treatment in bone preservation. Importantly, our murine studies indicate that B cell depletion has a direct effect on bone remodeling, primarily by reducing the osteoclastogenic signals, thus potentially diminishing bone resorption. This novel unrecognized effect should be taken into consideration when maintenance treatment is considered. MM and DN contributed equally to this work. Figure Disclosures No relevant conflicts of interest to declare.

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

With millions of new dental and orthopedic implants inserted annually, periprosthetic osteolysis becomes a major concern. In dentistry, peri-implantitis management includes cleaning using ultrasonic scaling. We examined whether ultrasonic scaling releases titanium particles and induces inflammation and osteolysis. Titanium discs with machined, sandblasted/acid-etched and sandblasted surfaces were subjected to ultrasonic scaling and we physically and chemically characterized the released particles. These particles induced a severe inflammatory response in macrophages and stimulated osteoclastogenesis. The number of released particles and their chemical composition and nanotopography had a significant effect on the inflammatory response. Sandblasted surfaces released the highest number of particles with the greatest nanoroughness properties. Particles from sandblasted/acid-etched discs induced a milder inflammatory response than those from sandblasted discs but a stronger inflammatory response than those from machined discs. Titanium particles were then embedded in fibrin membranes placed on mouse calvariae for 5 weeks. Using micro-CT, we observed that particles from sandblasted discs induced more osteolysis than those from sandblasted/acid-etched discs. In summary, ultrasonic scaling of titanium implants releases particles in a surface type-dependent manner and may aggravate peri-implantitis. Future studies should assess whether surface roughening affects the extent of released wear particles and aseptic loosening of orthopedic implants.

EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409–416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.

Ethanol (EtOH) withdrawal increases sensitivity to painful stimuli in adult rats. In this study, withdrawal from a single, acute administration of EtOH dose-dependently produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 (P7) rats. In contrast, P21 rats exhibited earlier and more prolonged mechanical allodynia but not thermal hyperalgesia. For both P7 and P21 rats, blood and spinal cord EtOH levels peaked at 30 minutes after administration, with P7 rats achieving overall higher spinal cord concentrations. Protein kinase C (PKC) has been implicated in mediating pain responses. Inhibitory PKCϵ− and γ-specific peptides attenuated mechanical allodynia and thermal hyperalgesia in P7 rats, whereas only the PKCγ inhibitor prevented mechanical allodynia in P21 rats. Immunoreactive PKCϵ in dorsal root ganglion and PKCγ in lumbar spinal cord increased at 6 hours after EtOH administration in P7 rats. In P21 rats, the density of PKCϵ immunoreactivity remained unchanged, whereas the density of PKCγ immunoreactivity increased and translocation occurred. These studies demonstrate developmental differences in neonatal nociceptive responses after withdrawal from acute EtOH and implicate a role for specific PKC isozymes in EtOH withdrawal–associated allodynia and hyperalgesia. Perspective This study examines age-specific nociceptive responses after ethanol exposure by using 2 different ages of rats. The results suggest that ethanol age-dependently alters sensitivity to mechanical and thermal stimuli via specific protein kinase C isozymes. These results begin to ascertain the mechanisms that produce abnormal pain after alcohol exposure.

Intracellularly acting peptide modulators of signaling enzymes provide a powerful means to regulate signaling events. Delivery of peptides into cells is facilitated by conjugation to carrier peptides, such as Tat. When peptides are irreversibly conjugated to Tat, Tat-mediated subcellular localization may predominate, resulting in mislocalization of the peptide cargo. We have used intracellularly acting peptides, conjugated to Tat by a disulfide bond, to modulate protein kinase C (PKC) signaling; these PKC-modulating peptides are released from Tat upon intracellular delivery. Previously, the distribution of these peptides within tissue and throughout the body had not been demonstrated. We show here intravascular delivery of a PKC-peptide, reversibly conjugated to Tat, resulted in distribution throughout cardiac tissue. In addition, a single injection resulted in selective modulation of PKC activity in many organs. Therefore, intracellularly acting peptide modulators of signaling enzymes, reversibly conjugated to Tat, have extensive biodistribution and can be used to modulate signaling pathways in vivo.

Background: Statins are lipid-lowering drugs with many beneficial pleiotropic effects. Cyclooxygenase (COX2) selective inhibitors that are commonly prescribed in orthopaedic patients may effect healing. Evidence indicates that statins stimulate COX2 activity. Hypothesis: Atorvastatin (ATV) administration will enhance tendon healing by stimulating the acute inflammatory phase via increasing the production of prostaglandin E2 (PGE2). Study Design: Controlled laboratory study. Methods: After experimental rotator cuff (RC) tearing and suturing, 48 Wistar rats were randomly allocated into 4 groups: (1) ATV (20 mg/kg), (2) celecoxib (CEL; COX2 inhibitor) (10 mg/kg), (3) ATV + CEL (20 mg/kg + 10 mg/kg), and (4) saline alone. Animals were sacrificed 3 weeks after RC tears and repair, and tendon integrity was tested biomechanically in tension. To further evaluate the underlying mechanism of action, human and rat primary tenocytes were obtained from the supraspinatus tendon. Cultures were treated with a therapeutic dosage of 5 commonly used statins: CEL, ATV + CEL, PGE2, and a selective antagonist of PGE2 receptor 4 (EP4). Cell proliferation (thymidine incorporation), migration (wound healing assay), and adhesion (iCELLigence) were evaluated. The expression of all PGE2 receptors (EPs) was determined by quantitative reverse transcription polymerase chain reaction. Results: Tension testing of healed tendons demonstrated significantly higher maximal loading and stiffness in the ATV group as compared with the saline (+30% and +20%, respectively; P < .001) and CEL groups (+33% and +50%, respectively; P < .005). Celecoxib alone did not affect tendon healing ( P = .88). In line with these in vivo results, tenocytes treated with statins demonstrated significantly higher proliferation rates; CEL abrogated this effect, and PGE2 treatment stimulated tenocyte proliferation even in the presence of CEL. Also, ATV stimulated the migration (wound healing) and adhesion of tenocytes. Among all PGE2 receptors, tenocytes mainly express EP4, and an EP4 selective antagonist blocked the effect of ATV. Conclusion: Results indicate that ATV enhances tendon healing by stimulating tenocyte proliferation, migration, and adhesion via increased COX2 activity and autocrine/paracrine PGE2 signaling. Findings also demonstrate that this effect is mediated by EP4 signaling. Clinical Relevance: Although chronic inflammation contributes to the development of tendinopathy, study results advocate for a positive role of PGE2 in tendon healing during the acute inflammatory phase that follows tendon surgical repair. It is therefore suggested that ATV should be further investigated as a possible modality to improve tendon healing.

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are in- creased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new thera- peutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO adminis- tration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology,andserummarkers,wefoundthatEPOinduced a32%-61%trabecularbonelosscausedby increasedbone resorption (+60%-88% osteoclast number) and reduced bone formation rate (219to274%; P

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, δPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of ɛPKC, δPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, δPKC and ɛPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of ɛPKC caused cardioprotection whereas activation of δPKC increased damage induced by ischemia in vitro and in vivo . In contrast, δPKC and ɛPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Abstract —Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [ r eceptors for a ctivated C k inase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.

The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCe gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCe (Tr-PKCe) mRNA, coding for N-terminal amino acids 1–116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCe protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCe, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCe and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCe grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCe were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCe in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCe mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.

Bone turnover is regulated by the coupled actions of osteoblasts, the bone-forming cells, and monocyte-derived osteoclasts, which mediate bone resorption. B cells were also shown to regulate bone metabolism, chiefly via paracrine signals. Depending on their state and/or mode of activation, B cells may inhibit or enhance osteoclastogenesis. In mammals, B cell development and maturation occurs in the bone marrow (BM), spleen, and other peripheral lymphoid tissues. In the BM, Pro-B cells sequentially differentiate into Pre-B and immature B cells. Whether BM B cells can transdifferentiate into osteoclasts remains controversial, since osteoclast differentiation from residual monocytic precursors in the cultures was not excluded in the earlier studies. Osteoclasts and B cells arise from distinct myeloid and lymphoid progenitors, respectively, which are downstream of a common multipotent progenitor cell. Monocyte differentiation into osteoclasts relies on monocyte-macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). Here, we investigated the possibility that BM B-cells contribute to bone loss by transdifferentiating into bone-resorbing osteoclasts. We found that B220+CD19+ cells can transdifferentiate into multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the presence of RANKL and M-CSF. Our results show that Pro-B cells (B220+CD19+CD43HighIGM-), but not Pre-B cells (B220+CD19+CD43LowIGM-), nor immature B cells (B220+CD19+CD43-IGM+), could transdifferentiate into osteoclasts (16%±3.7 vs. 0.79%±0.28 and 0.48%±0.13 osteoclasts area, respectively). Moreover, among the Pro-B cells, only those expressing M-CSF receptor (CD115) could transdifferentiate into functional osteoclasts (18%±6.55 vs. 0.11%±0.05 osteoclasts area, respectively, Figure 1A and B). To unequivocally establish the generation of osteoclasts from B-cells, we next utilized a mouse model in which all B cell lineage-derived progenies express EYFP. We found that B cells isolated from BM of CD19-Cre:EYFP mice differentiated into TRAP+ multinucleated osteoclasts that were also positive for EYFP (Figure1C). Erythropoietin (EPO) is a crucial kidney-derived hormone responsible for erythropoiesis. Once thought to act solely on the erythroid compartment to potentiate red blood cell production, it became evident that EPO receptors are also found on the monocytic lineage (monocytes, macrophages and dendritic cells). In that respect, we have reported that EPO directly stimulates bone loss via activation of EPO-R signaling in the monocytic lineage (Hiram-Bab et al., 2015). Here we report that B cells express EPO-R, and that EPO enhances Pro-B cell differentiation into osteoclasts by 70% (p Conceivably, in other scenarios, e.g., sex hormone deficiency, certain hematological cancers, and treatment with anti-CD20, increased B cell lymphocytes contribute to the bone loss phenotype due to transdifferentiation of B-cell precursors into osteoclasts. Taken together, our data suggest a new physio-pathological role for BM B cell precursors in bone metabolism, via their capacity to differentiate into functional osteoclasts, and a possible role for EPO in this process. Figure 1 Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220+CD19+CD43HighIgM-CD115+). Right - Pro-B cells not expressing CD115 (B220+CD19+CD43HighIgM-CD115-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p Disclosures No relevant conflicts of interest to declare.

Background and rationale: Identifying risk factors for bone loss is of utmost importance in the prevention of osteoporotic bone fractures. We and others have recently described the dramatic erythropoietin (EPO)-induced bone loss. Recombinant EPO and its long-acting derivative, darbepoetin alpha (DA), are frequently and interchangeably used in clinical practice for similar indications including diseases (e.g. multiple myeloma) in which bone mass may be compromised already, as a part of the disease process. Here, we examined in a murine model, the dose response to the erythroid stimulating agents (ESA) EPO and DA, in the erythroid and bone compartments. Methods:C57B6/JRcchsd 9-week old female mice were treated for two weeks with either EPO or DA using two dose intensities. EPO and DA were administered subcutaneously thrice or once weekly, respectively, for a total weekly dose of 27IU/mouse or 540IU/mouse, hereafter defined correspondingly as intermediate (ID) and high dose (HD) subgroups. Bone morphometry of the distal femoral metaphysis was assessed using microCT. Flow cytometry was used for immunophenotyping of bone marrow (BM) cells. One-way ANOVA was used to compare variables among the treatment groups. Results: First, we found that ID-DA induced bone loss to a similar extent as ID-EPO as reflected by a significant (p0.05 for EPO versus DA groups). In view of the established role of B cells in bone metabolism, we determined the effects of EPO and DA on BM B cell compartment. HD-EPO but not ID-EPO induced a "maturation arrest"-like state in the bone marrow B cell population, increasing the proportion of B220+/CD19-/CD43high/IgM- pre-pro-B two-fold and B220+/CD19+/CD43high/IgM- pro-B cells 2.8-fold (p Conclusion:Applying clinically comparable dosing regimen, both recombinant EPO and DA exert a similar, dose-dependent bone loss, not directly proportional to the erythroid response. However, DA, in contrast to EPO, spares the preosteoblast and bone marrow B cell populations. These finding suggest a different mechanism of action of these closely related ESA preparations on the skeletal and immune systems thus warranting further exploration. Finally, our findings advocate for initiating and maintaining patients with the lowest possible doses of EPO/DA to minimize untoward skeletal and other extra-erythropoietic effects, while achieving the desired correction of anemia. This study was supported by a grant from the Israel Cancer Association to DN, YG and MM. Disclosures No relevant conflicts of interest to declare.

We have previously shown that domains involved in binding of protein kinase C (PKC) isozymes to their respective anchoring proteins (RACKs) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named psiRACK peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the psiRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the psiRACK sequence. We focused on epsilonPKC psiRACK peptide, where the pseudo-epsilonRACK sequence (psiepsilonRACK; HDAPIGYD; corresponding to epsilonPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to epsilonRACK285-292) in the second amino acid. Here we show that changing the charge of the psiepsilonRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.

Alzheimer’s disease (AD) is characterized by extracellular deposits of amyloid beta peptide (Aβ), a peptide that is generated upon proteolytic cleavage of amyloid precursor protein (APP). The events leading to the development of AD and their sequence are not yet fully understood. Protein kinase C (PKC) has been suggested to have a significant role in controlling neuronal degeneration and in the aberrant signal transduction taking place in AD. Several studies document a deficit in PKC levels and activity in brains of AD patients when compared with those of normal controls. Such a decrease in PKC could have serious implications since certain PKC isozymes were shown to drive the APP proteolytic cleavage into a non-amyloidogenic pathway. Reduced levels of distinct PKC isozymes could thus contribute to driving APP processing toward an amyloidogenic pathway. The direct cause for the down-regulation of PKC in AD brains is still unknown. In that respect, we tested in this study whether APP may play a role in PKC reduction. We show in three different cell lines (CHO, COS and BOSC) that overexpression of APP leads to decreased PKC levels. This decrease was found to be specific for the epsilon PKC isozyme whereas the levels of delta, alpha and conventional PKC remained unchanged. Furthermore, we observed this decrease for both active, membrane-associated and inactive, cytosolic epsilon PKC. APP-driven decrease in epsilon PKC is most likely mediated by a factor in the culture medium, since transfer of medium from cultured cells overexpressing APP to naive, non-overexpressing cells, has also led to the selective decrease in epsilon PKC levels. Taken together, our results suggest that APP expression levels may play a role in the decrease of epsilon PKC levels in AD brains and could thus affect the responsiveness of AD brain tissues to growth factors and neurotransmitters.

Ethanol (EtOH) withdrawal increases sensitivity to painful stimuli in adult rats. In this study, withdrawal from a single, acute administration of EtOH dose-dependently produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 (P7) rats. In contrast, P21 rats exhibited earlier and more prolonged mechanical allodynia but not thermal hyperalgesia. For both P7 and P21 rats, blood and spinal cord EtOH levels peaked at 30 minutes after administration, with P7 rats achieving overall higher spinal cord concentrations. Protein kinase C (PKC) has been implicated in mediating pain responses. Inhibitory PKC- and gamma-specific peptides attenuated mechanical allodynia and thermal hyperalgesia in P7 rats, whereas only the PKCgamma inhibitor prevented mechanical allodynia in P21 rats. Immunoreactive PKC in dorsal root ganglion and PKCgamma in lumbar spinal cord increased at 6 hours after EtOH administration in P7 rats. In P21 rats, the density of PKC immunoreactivity remained unchanged, whereas the density of PKCgamma immunoreactivity increased and translocation occurred. These studies demonstrate developmental differences in neonatal nociceptive responses after withdrawal from acute EtOH and implicate a role for specific PKC isozymes in EtOH withdrawal-associated allodynia and hyperalgesia.This study examines age-specific nociceptive responses after ethanol exposure by using 2 different ages of rats. The results suggest that ethanol age-dependently alters sensitivity to mechanical and thermal stimuli via specific protein kinase C isozymes. These results begin to ascertain the mechanisms that produce abnormal pain after alcohol exposure.

Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription – polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (Km= 51.3 ± 12.9 μM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (Bmax= 520 ± 10 fmol/mg protein, Kd= 0.11 ± 0.04 nM, 1.6 × 105NBTI-binding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.Key words: adenosine, nucleoside transport, HL-1 cells, cardiovascular, glucose transport, protein kinase C.

PKC isozymes are the major binding proteins for tumor-promoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKCalpha at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKCepsilon was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of PKCepsilon or alpha. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKCalpha 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKCepsilon. However, 8 of 11 PCs had major isozyme-specific reductions of the PKCepsilon protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKCepsilon are highly prevalent in thyroid tumors and may play a significant role in their development.

The negative effect of hypoxia on bone metabolism is well established but its mechanism of action is not fully understood. Hypoxia triggers the production of erythropoietin (EPO), a hormone most recognized for its hematopoietic function. An increasing number of roles unrelated to red blood cell production have been attributed to EPO, many of which are mediated by non-erythroid cells. In light of the controversy on the effect of increased serum levels of EPO on the skeleton, we investigated here the effect of the hormone on bone metabolism using EPO overexpressing (Tg6), and EPO-administered adult mice. In these models, the increase in EPO levels is similar to its physiologic increase at high altitude without the confounding direct effect of hypoxia on bone cells. Using microcomputed tomography, histology and serum markers we found that high EPO levels result in a severe trabecular bone loss (-32 to -61% decrease in bone density in Tg6 and EPO-injected animals, respectively; p EPO has direct stimulatory effects in vitro on both macrophages and preosteoclasts thus coupling immune and skeletal systems, Activation of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of 5T33 multiple myeloma cells. EPO strongly stimulated osteoclastogenesis (TRAP staining, Figure 1B) and pit resorption (measured in calcium-phosphate-coated wells) in a cell-autonomous manner. Furthermore, our data indicate that EPO receptor (EPO-R) signaling in osteoclast precursors involves the Jak2 and PI3K pathways, but is independent of the MAPK/MEK pathway. In addition to the direct effect of EPO on monocyte derived cells, high EPO also led to a 1.6 fold (p=0.03) increase in the transcript levels of the osteoclastogenic cytokine RANKL in total bone marrow. Notably, the major sources of RANKL are B cells and osteoblasts, and our data indicate that both cell types express EPO-R, suggesting that they are direct targets of EPO. Accordingly we found an EPO-related increase in membrane bound RANKL on B cells (1.8-fold, p=0.0003) and osteoblasts (1.16-fold, p=0.001) isolated from bone marrow (Figure 1C). To test whether EPO-R on osteoblasts also mediates the attenuation of bone formation, we treated isolated osteoblasts with EPO. However, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, as demonstrated by RT-qPCR of marker genes and mineralization assays. Taken together, these findings demonstrate that EPO strongly regulates bone mass by stimulating osteoclastogenesis and attenuating bone formation. The osteoclastogenic action of EPO is mediated both directly by EPO-R on the monocytic lineage and indirectly via EPO-R signaling on B cells and osteoblasts. Regarding bone formation, the negative effect of EPO on bone formation rate in vivo was not reproduced in isolated cells, thus implying the involvement of cells beyond the osteoblastic lineage. We also propose a new mechanism for hypoxia-induced bone loss that involves EPO action on monocytic, B cell and osteoblastic lineages. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). epsilon Protein kinase C (epsilonPKC) has been suggested to mediate preconditioning. Here, we describe an epsilonPKC-selective agonist octapeptide, psiepsilon receptor for activated C-kinase (psiepsilonRACK), derived from an epsilonPKC sequence homologous to its anchoring protein, epsilonRACK. Introduction of psiepsilonRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased epsilonPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that epsilonPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this epsilonPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation.