Anthracycline cardiotoxicity is exacerbated by global p38ß genetic ablation in a sexually dimorphic manner but unaltered by cardiomyocyte-specific p38α loss.

Severe cardiotoxic effects limit the efficacy of doxorubicin (DOX) as a chemotherapeutic agent. Activation of intracellular stress signaling networks, including p38 mitogen-activated protein kinase (MAPK), has been implicated in DOX-induced cardiotoxicity (DIC). However, the roles of the individual p38 isoforms in DIC remain incompletely elucidated. We recently reported that global p38δ deletion protected female but not male mice from DIC, whereas global p38γ deletion did not significantly modulate it. Here we studied the in vivo roles of p38α and p38ß in acute DIC. Male and female mice with cardiomyocyte-specific deletion of p38α or global deletion of p38ß and their wild-type counterparts were injected with DOX. Survival and health were tracked for 10 days postinjection. Cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picrosirius red staining. Expression and activation of signaling proteins and inflammatory markers were measured by Western blot, phosphorylation array, and chemokine/cytokine array. Global p38ß deletion significantly aggravated DIC and worsened cardiac electrical and mechanical function deterioration in female mice. Mechanistically, DIC in p38ß-null female mice correlated with increased autophagy, sustained hyperactivation of proapoptotic JNK signaling, as well as remodeling of a myocardial inflammatory environment. In contrast, cardiomyocyte-specific deletion of p38α improved survival of DOX30-treated male mice 5 days posttreatment but did not influence cardiac function in DOX-treated male or female mice. Our data highlight the sex- and isoform-specific roles of p38α and p38ß MAPKs in DOX-induced cardiac injury and suggest a novel in vivo function of p38ß in protecting female mice from DIC.NEW & NOTEWORTHY We show that p38α and p38ß have distinct in vivo functions in a murine model of acute DIC. Specifically, although conditional cardiomyocyte-specific p38α deletion exhibited mild cardioprotective effects in male mice, p38ß deletion exacerbated the DOX cardiotoxicity in female mice. Our findings caution against employing pyridinyl imidazole inhibitors that target both p38α and p38ß isoforms as a cardioprotective strategy against DIC. Such an approach could have undesirable sex-dependent effects, including attenuating p38ß-dependent cardioprotection in females.

Open thoracic surgical implantation of cardiac pacemakers in rats.

Genetic engineering and implantable bioelectronics have transformed investigations of cardiovascular physiology and disease. However, the two approaches have been difficult to combine in the same species: genetic engineering is applied primarily in rodents, and implantable devices generally require larger animal models. We recently developed several miniature cardiac bioelectronic devices suitable for mice and rats to enable the advantages of molecular tools and implantable devices to be combined. Successful implementation of these device-enabled studies requires microsurgery approaches that reliably interface bioelectronics to the beating heart with minimal disruption to native physiology. Here we describe how to perform an open thoracic surgical technique for epicardial implantation of wireless cardiac pacemakers in adult rats that has lower mortality than transvenous implantation approaches. In addition, we provide the methodology for a full biocompatibility assessment of the physiological response to the implanted device. The surgical implantation procedure takes ~40 min for operators experienced in microsurgery to complete, and six to eight surgeries can be completed in 1 d. Implanted pacemakers provide programmed electrical stimulation for over 1 month. This protocol has broad applications to harness implantable bioelectronics to enable fully conscious in vivo studies of cardiovascular physiology in transgenic rodent disease models.

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models.

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients' care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

Fully implantable and bioresorbable cardiac pacemakers without leads or batteries.

Temporary cardiac pacemakers used in periods of need during surgical recovery involve percutaneous leads and externalized hardware that carry risks of infection, constrain patient mobility and may damage the heart during lead removal. Here we report a leadless, battery-free, fully implantable cardiac pacemaker for postoperative control of cardiac rate and rhythm that undergoes complete dissolution and clearance by natural biological processes after a defined operating timeframe. We show that these devices provide effective pacing of hearts of various sizes in mouse, rat, rabbit, canine and human cardiac models, with tailored geometries and operation timescales, powered by wireless energy transfer. This approach overcomes key disadvantages of traditional temporary pacing devices and may serve as the basis for the next generation of postoperative temporary pacing technology.

p38δ genetic ablation protects female mice from anthracycline cardiotoxicity.

The efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38γ and p38δ, remain uncharacterized. We aimed to determine the potential cardioprotective effects of p38γ and p38δ genetic deletion in mice subjected to acute DOX treatment. Male and female wild-type (WT), p38γ-/-, p38δ-/-, and p38γ-/-δ-/- mice were injected with 30 mg/kg DOX and their survival was tracked for 10 days. During this period, cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picro Sirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy. Significantly improved survival was observed in p38δ-/- female mice post-DOX relative to WT females, but not in p38γ-/- or p38γ-/-δ-/- male or female mice. The improved survival in DOX-treated p38δ-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as suggested by increased LC3-II level, and decreased mammalian target of rapamycin activation was also observed in DOX-treated p38δ-/- females. p38δ plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38δ targeting could be a potential cardioprotective strategy in anthracycline chemotherapy.NEW & NOTEWORTHY This study for the first time identifies the sex-specific roles of the alternative p38γ and p38δ MAPK isoforms in promoting doxorubicin (DOX) cardiotoxicity. We show that p38δ and p38γ/δ systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38δ-/- females and autophagy marker was increased.

Cell Type-Specific p38δ Targeting Reveals a Context-, Stage-, and Sex-Dependent Regulation of Skin Carcinogenesis.

Activation and/or upregulated expression of p38δ are demonstrated in human skin malignancies including cutaneous squamous cell carcinoma, suggesting a role for p38δ in skin carcinogenesis. We previously reported that mice with germline deletion of the p38δ gene are significantly protected from chemical skin carcinogenesis. Here, we investigated the effects of cell-selective targeted ablation of p38δ in keratinocytes and in immune (myeloid) cells on skin tumor development in a two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical mouse skin carcinogenesis model. Conditional keratinocyte-specific p38δ ablation (p38δ-cKO∆K) did not influence the latency, incidence, or multiplicity of chemically-induced skin tumors, but led to increased tumor volume in females during the TPA promotion stage, and reduced malignant progression in males and females relative to their wild-type counterparts. In contrast, conditional myeloid cell-specific p38δ deletion (p38δ-cKO∆M) inhibited DMBA/TPA-induced skin tumorigenesis in male but not female mice. Thus, tumor onset was delayed, and tumor incidence, multiplicity, and volume were reduced in p38δ-cKO∆M males compared with control wild-type males. Moreover, the percentage of male mice with malignant tumors was decreased in the p38δ-cKO∆M group relative to their wild-type counterparts. Collectively, these results reveal that cell-specific p38δ targeting modifies susceptibility to chemical skin carcinogenesis in a context-, stage-, and sex-specific manner.

Managing Seborrheic Keratosis: Evolving Strategies and Optimal Therapeutic Outcomes.

Seborrheic keratosis (SK) is the most common skin tumor seen by dermatologists in everyday practice. Although the lesions are mostly benign, many patients still elect to have asymptomatic SK removed. The historical standards of treatment are cryosurgery and electrocautery, two surgical options that are effective at lesion removal but have high rates of postoperative adverse events such as treatment-site scarring and pigmentary alterations. The cosmetic outcomes of SK treatment modalities are of keen interest to dermatologists, as the American population becomes increasingly more diverse. In this article, the inclusion of darker Fitzpatrick skin types into clinical studies investigating post-treatment side effects of SK therapy is reviewed. The recent approval of a 40% hydrogen peroxide topical formulation is discussed in light of these issues, and several non-invasive topical treatments that optimize cosmetic outcomes of SK lesion removal are highlighted. Finally, treatment strategies aimed at reducing cost and minimizing the burden of adverse sequelae are provided. J Drugs Dermatol. 2018;17(9):933-940.

Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin.

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1ß, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment.

HCV genome-wide genetic analyses in context of disease progression and hepatocellular carcinoma.

Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV's sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.

Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice.

Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions.

Serum response factor controls transcriptional network regulating epidermal function and hair follicle morphogenesis.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.

Sexual transmission of hepatitis C virus among monogamous heterosexual couples: the HCV partners study.

UNLABELLED: The efficiency of hepatitis C virus (HCV) transmission by sexual activity remains controversial. We conducted a cross-sectional study of HCV-positive subjects and their partners to estimate the risk for HCV infection among monogamous heterosexual couples. A total of 500 anti-HCV-positive, human immunodeficiency virus-negative index subjects and their long-term heterosexual partners were studied. Couples were interviewed separately for lifetime risk factors for HCV infection, within-couple sexual practices, and sharing of personal grooming items. Blood samples were tested for anti-HCV, HCV RNA, and HCV genotype and serotype. Sequencing and phylogenetic analysis determined the relatedness of virus isolates among genotype-concordant couples. The majority of HCV-positive index subjects were non-Hispanic white, with a median age of 49 years (range, 26-79 years) and median of 15 years (range, 2-52 years) of sexual activity with their partners. Overall, HCV prevalence among partners was 4% (n=20), and nine couples had concordant genotype/serotype. Viral isolates in three couples (0.6%) were highly related, consistent with transmission of virus within the couple. Based on 8,377 person-years of follow-up, the maximum incidence rate of HCV transmission by sex was 0.07% per year (95% confidence interval, 0.01-0.13) or approximately one per 190,000 sexual contacts. No specific sexual practices were related to HCV positivity among couples. CONCLUSION: The results of this study provide quantifiable risk information for counseling long-term monogamous heterosexual couples in which one partner has chronic HCV infection. In addition to the extremely low estimated risk for HCV infection in sexual partners, the lack of association with specific sexual practices provides unambiguous and reassuring counseling messages.

Morphine modulation of thrombospondin levels in astrocytes and its implications for neurite outgrowth and synapse formation.

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple µ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by µ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFß1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of µ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.

Kappa opioids promote the proliferation of astrocytes via Gbetagamma and beta-arrestin 2-dependent MAPK-mediated pathways.

GTP binding regulatory protein (G protein)-coupled receptors can activate MAPK pathways via G protein-dependent and -independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and beta-arrestin 2 pathways in kappa opioid receptor-induced, extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non-nitrogenous agonist, C(2)-methoxymethyl salvinorin B (MOM-Sal-B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gbetagamma subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of beta-arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM-Sal-B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM-Sal-B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gbetagamma subunits or beta-arrestin 2, suggesting that both G protein-dependent and -independent ERK pathways are required for this outcome.

p120-catenin is a key component of the cadherin-gamma-secretase supercomplex.

In this work, we show several previously unknown features of p120-catenin in a cadherin-catenin complex that are critical for our understanding of cadherin-based adhesion and signaling. We show that in human epithelial A-431 cells, nearly all p120 molecules engage in high-affinity interaction with E-cadherin-catenin complexes located at the cellular surface. p120 is positioned in proximity to alpha-catenin in the complex with cadherin. These findings suggest a functional cooperation between p120 and alpha-catenin in cadherin-based adhesion. A low level of cadherin-free p120 molecules, in contrast, could facilitate p120-dependent signaling. Finally, we present compelling evidence that p120 is a key linker cementing the E-cadherin-catenin complex with the transmembrane protease gamma-secretase. The cell-cell contact location of this supercomplex makes it an important candidate for conducting different signals that rely on gamma-secretase proteolytic activity.

SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo.

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors.

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes.

Acute mu and kappa opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The mu agonist, [D-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas kappa agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant mu opioid receptor that binds CaM poorly or a dominant negative mutant of PKCepsilon were used as a model system to study mu signaling. Evidence was gained to implicate CaM and PKCepsilon in DAMGO stimulation of ERK. DAMGO activation of PKCepsilon and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the mu receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCepsilon. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCzeta, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCzeta. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic mu and kappa opioids.