Phorbol 12-myristate 13-acetate Response Research Articles

Abstract 1431: Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines

Abstract Oncogenic mutations in the NRAS gene are found in many cancers including a subset of acute myeloid leukemias (AML). Established AML cell lines exhibiting hyperactive signaling through the RAS-RAF-MEK-ERK pathway show constitutive ERK activation generating a transcriptome that mediates escape from programmed cell death and continuous proliferation. It has been established that exposure of many AML cell lines to phorbol esters such as phorbol-12-myristate-13-acetate (PMA) mediates cell cycle arrest, myeloid differentiation, and apoptosis. This effect seems counterintuitive given that PMA irreversibly activates protein kinase C (PKC), which enhances ERK activity leading to transcriptional upregulation of target genes controlled by ERK activated transcription factors, many of which promote proliferation. Here we define a set of PMA response genes in AML cell lines driven by oncogenic NRAS that encode negative regulators of RAS-RAF-MEK-ERK signaling, including SPRY2, RASA1, DUSP 5, and DUSP6. Additionally, we show that overexpression of SPRY2 alone attenuates proliferation by inhibiting ERK activation and altering the AML cell transcriptome through the activation of genes encoding members of the AP-1 transcription factor family, FOS, JUNB, and JUND, and the cyclin-CDK inhibitors CDKN1A and CDKN2B. We hypothesize that oncogenic NRAS signaling is overridden by PMA treatment through the activation of ERK target genes that ultimately inhibit the MAPK-ERK pathway, such as SPRY2. Finally, we show that SPRY 2 expression in AML patient samples represents a survival prognostic indicator where higher expression levels correlate with improved overall survival. Citation Format: Michael Roberts, Sher Bahadur, Coltin Albitz, Jae Chung, Samantha Garcia, Cuong Nguyen, Sophia Kovatsis. Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1431.

Multiple, Independent T Cell Lymphomas Arising in an Experimentally FIV-Infected Cat during the Terminal Stage of Infection.

Our laboratory has serially reported on the virologic and immunopathologic features of a cohort of experimental feline immunodeficiency virus (FIV)-infected cats for more than eight years. At 8.09 years post infection (PI), one of these animals entered the terminal stage of infection, characterized by undulating hyperthermia, progressive anorexia, weight loss, and pancytopenia; the animal was not responsive to therapeutic interventions, necessitating euthanasia six weeks later (8.20 years PI). Subsequent analyses indicated that neoplastic lymphocytes infiltrated multiple cervical lymph nodes and a band-like region of the mucosal lamina propria within a segment of the intestine. Immunohistochemistry and T cell clonality testing determined that the nodal and intestinal lesions were independently arising from CD3 T cell lymphomas. In-situ RNA hybridization studies indicated that diffuse neoplastic lymphocytes from the cervical lymph node contained abundant viral nucleic acid, while viral nucleic acid was not detectable in lymphocytes from the intestinal lymphoma lesion. The proviral long terminal repeat (LTR) was amplified and sequenced from multiple anatomic sites, and a common clone containing a single nucleotide polymorphism was determined to be defective in response to phorbol myristate acetate (PMA)-mediated promoter activation in a reporter gene assay. This assay revealed a previously unidentified PMA response element within the FIV U3 region 3’ to the TATA box. The possible implications of these results on FIV-lymphoma pathogenesis are discussed.

Abstract 3815: Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogues in LNCaP and U937 cancer cell lines provides insight into their differential mechanism of action

Abstract Bryostatin 1 has attracted great interest as a cancer chemotherapeutic agent with a unique mechanism of action. Whereas bryostatin 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. We wish to understand its mechanism(s) of action and how the newly emerging synthetic bryostatin analogs functionally resemble or differ from bryostatin 1. Previously, we have compared patterns of biological response to bryostatin 1, the phorbol ester phorbol 12-myristate 13-acetate (PMA), and the synthetic bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical phorbol ester biological response in either cell line, whereas the bryostatin analog Merle 23 resembles PMA in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared patterns of transcriptional response to bryostatin 1, PMA, and Merle 23 in the same two human cancer cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes highly regulated downstream of protein kinase C. In both cell lines, bryostatin 1 differed from phorbol ester primarily in having a shorter duration of transcriptional modulation. In LNCaP cells bryostatin 1 induced the majority of the genes similarly to PMA at 2 hours but to much lower levels at 6, 12, and 24 hrs. In U937 cells the bryostatin 1 induced response was similar to that of PMA at 2 hrs but progressively less than that of PMA at 8 hrs and 24 hrs. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the PMA response at these later times. A notable difference between the two cell lines was in the extent of transcriptional response to PMA, which was much greater in the LNCaP cells than in the U937 cells. In both cell lines Merle 23 induced a pattern of transcription largely like that of PMA rather than bryostatin 1 although with a modest reduction in expression at later times (12 and 24 hrs) in the LNCaP cells. We thus conclude that the difference in biological response of the two cell lines to Merle 23 most likely does not reflect an underlying difference in mechanism but rather reflects the greater absolute magnitude of the differences in response in the LNCaP cells compared to the U937 cells. For a series of bryostatins and synthetic analogues which ranged from bryostatin 1-like to phorbol ester-like in activity on the U937 cells for proliferation and differentiation, the duration of transcriptional response correlated with their pattern of biological activity, suggesting that this may provide a useful endpoint for structure activity analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3815. doi:1538-7445.AM2012-3815

Abstract 4058: Diverse patterns of biological response to phorbol esters and related protein kinaseC activators in LNCaP human prostate cancer cells

Abstract All protein kinase C activators are not equivalent. Whereas phorbol 12-myristate 13-acetate (PMA) is the paradigmatic tumor promoter, bryostatin 1 or ingenol 3-angelate are in clinical trials as cancer chemotherapeutic agents. To better understand the structural features contributing to different biological outcomes, we tested a series of phorbol esters differing widely in hydrophobicity in two systems in which bryostatin 1 and PMA give very different responses. With U937 human leukemia cells, PMA inhibits cell growth and induces cell attachment, unlike bryostatin 1. We found that all the phorbol derivatives, like PMA, inhibited growth in a dose dependent manner. They likewise all induced attachment, a measure of differentiation, but several (sapintoxin D, phorbol 12, 13-dibenzoate, phorbol 12, 13-dihexanoate) differed in displaying a biphasic curve for this latter response. In the LNCaP cells, PMA inhibits growth and induces tumor necrosis factor alpha (TNF-alpha) secretion, whereas bryostatin 1 does not. We found that inhibition of growth in response to the phorbol esters ranged from full to partial (31-100 % of the PMA response) and the dose response curves ranged from monophasic to steeply biphasic. Similar divergent behavior was observed for induction of TNF-alpha secretion. For example, the secretion induced by phorbol 12, 13-dibenzoate and phorbol 12, 13-didecanoate was only 22% and 40% of the maximal response, respectively, and the response induced by sapintoxin D was very biphasic: 10 and 30 nM drug induced 56-59%, and 3000 and 10,000 nM induced only 8% and 5% of the maximal response. While none of the compounds induced as little response as did bryostatin 1, the results suggest that the difference was more quantitative in this system than qualitative. We conclude, moreover, that the hydrophobicity of the compounds was not the critical determinant of activity. Unlike the biological response, the translocation pattern of GFP-PKC delta did correlate with hydrophobicity, as the more lipophilic compounds PMA, octylindolactam V and phorbol 12, 13-didecanoate induced translocation to the plasma membrane, followed by translocation to internal membranes, while the more hydrophilic compounds translocated GFP-PKC delta mostly to the internal membranes. We thus conclude that the overall pattern of PKC delta translocation by itself cannot predict the different responses observed. Finally, Nano-pro technology, which can fingerprint the phosphorylation pattern of a protein, is revealing that the different phorbol esters induce divergent phosphorylation patterns of PKC delta, the isoform largely responsible for the phorbol ester induced apoptosis and TNF-alpha secretion in the LNCaP cells, and may provide a powerful tool for viewing the integrated outcome of the numerous elements impinging on this kinase following ligand interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2011-4058

Phorbol ester-induced human cytomegalovirus major immediate-early (MIE) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells.

The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE(+)) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-kappaB (kappaB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and kappaB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-kappaB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and kappaB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-kappaB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

Identification of a minimal cis-element and cognate trans-factor(s) required for induction of Rac2 gene expression during K562 cell differentiation

Rac2 is a Rho family GTPase that is widely expressed in hematopoietic cells and plays a critical role in host defense. This study investigates the mechanisms responsible for increased Rac2 gene expression during myeloid cell differentiation. Treatment of K562 chronic myelogenous leukemia cells with phorbol-12-myristate-13-acetate (PMA) induces megakaryocytic differentiation and Rac2 gene transcription following a lag of 6–12 h. Promoter/luciferase reporter gene assays reveal that a 135 bp cis-element located between − 4223 and − 4008 bp upstream of the Rac2 transcription start site is necessary and sufficient for PMA-induced gene expression. The AP1 transcription factor binds to three cis-elements within the 135 bp Rac2 gene regulatory region both in vitro and in vivo following PMA treatment, and mutagenesis of the AP1 binding sites ablates the PMA responsiveness of the 135 bp Rac2 gene regulatory region. Over-expression of AP1 is sufficient to induce expression of a transiently transfected Rac2 promoter/luciferase plasmid, but not the endogenous Rac2 gene. Induction of AP1 in vitro DNA-binding activity is apparent within 1 h of PMA stimulation. However, AP1 binding to the endogenous Rac2 promoter exhibits a lag of 5–9 h, which correlates with reduced histone H3-Lys9 methylation, increased histone H3 acetylation, and increased nuclease accessibility within the 135 bp Rac2 gene regulatory region. These results demonstrate that PMA induction of Rac2 expression during terminal myeloid differentiation requires the coordinate induction of transcription factors and remodeling of Rac2 gene chromatin structure.

Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

Galphaq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Galphaq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5' upstream region of the Galphaq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5' upstream of the Galphaq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Galphaq 5' upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt -1042/-1037 eliminated the difference. Gel-shift studies on Galphaq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence -1042/-1037. Silencing of endogenous EGR-1 inhibited Galphaq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence -1042/-1037 bp in Galphaq promoter mediates the induction of Galphaq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Galphaq promoter, and new insights into regulation of Galphaq gene by EGR-1.

Phorbol Ester Stimulates Corticotropin-Releasing Hormone Gene Promoter Activity Through a cAMP Regulatory Element in Primary Placental Cells

Placental corticotropin-releasing hormone (CRH) plays an important role in the mechanisms controlling human pregnancy and parturition, and several endogenous factors are known to regulate placental CRH gene expression. In this article, the authors investigate the regulation of the CRH gene's promoter activity by a protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), in primary cultures of placental cells. The PMA stimulation of the CRH gene promoter activity was dose dependent, and further studies, including progressive deletion and mutation analysis of the CRH promoter, localized the region essential for PMA responsiveness to a consensus cyclic adenosine monophosphate regulatory element (CRE). Furthermore, estradiol treatment resulted in decreases of both basal and PMA-stimulated promoter activity when the CRE element was present but had no effect when the CRE element was absent. Thus, PMA stimulates CRH gene transcriptional activity through the CRE, suggesting that cross-talk between PKC and protein kinase A signaling pathways targets this regulatory element in placental cells.

Neutrophil dysfunction after biomaterial contact in an in vitro model of cardiopulmonary bypass

Cardiopulmonary bypass (CPB) induces neutrophil degranulation and superoxide anion production in vivo. We hypothesized that CPB-associated neutrophil dysregulation alters neutrophil adhesion to vascular endothelial cells and the extracellular matrix. We, therefore, recirculated neutrophils in polyvinyl chloride (PVC) tubing using a roller pump model and thereafter measured adhesion to cultured microvascular endothelial cells and gelatin-coated surfaces. Recirculation-induced neutrophil priming or exhaustion was tested by boosting with phorbol myristate-acetate (PMA) or N-formyl-methiolyl-leucyl-phenylalanine (FMLP) before quantification of adhesion. After recirculation, neutrophils retained their adhesive capability to vascular endothelial cells, whereas adhesion to gelatin increased. This increase was not seen when the neutrophils were recirculated with a rotator instead of a roller pump, indicating that not only the pump mode but also foreign surface contact was of significance. The neutrophil PMA response after recirculation was not altered compared to resting neutrophils prestimulated with PMA. Recirculated neutrophils adhered less to cultured vascular endothelial cells after FMLP activation and more to gelatin compared to resting neutrophils prestimulated with FMLP. It is conceivable that dysregulation of neutrophil adhesive capability may play a part in the development of tissue damage after CPB.

Inhibitory Role of Ser-425 of the α1 2.2 Subunit in the Enhancement of Cav 2.2 Currents by Phorbol-12-myristate, 13-Acetate

Voltage-gated calcium channels (Ca(v)) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Ca(v) 2.3 currents are increased by both PMA and acetyl-beta-methylcholine (MCh). MCh-selective sites were identified in the alpha(1) 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102-4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559-23565). The hypothesis that PMA sites in the alpha(1) 2.2 subunit are homologous to the PMA-responsive sites in alpha(1) 2.3 subunit was tested with Ser/Thr --> Ala mutations in the alpha(1) 2.2 subunit. WT alpha(1) 2.2 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits. Inward current (I(Ba)) was recorded using Ba(2+) as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites in alpha(1) 2.2 and alpha(1) 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the alpha(1) 2.2 subunit.

Role of RasGRP in Phorbol Ester Response in EL4 Lymphoma Cells

EL4, a murine thymoma cell line, exists in phorbol ester-sensitive, resistant, and intermediate variants. Sensitive cells respond to phorbol 12-myristate 13-acetate (PMA) with robust activation of Erk MAPKs. In contrast, in resistant cells, Erk activation is weak, delayed, and requires higher concentrations of PMA. Intermediate cells exhibit partial PMA responsiveness. We hypothesized that PMA-induced Erk activation in EL4 cell lines is mediated by RasGRP (Ras guanyl releasing protein), a phorbol ester binding protein that regulates activation of Ras. Ras activation was assessed by a Ras-GTP pulldown assay, while Erk activation was tested by immunoblotting with anti-phospho-Erk. Our studies showed that RasGRP protein is expressed in PMA-sensitive cells, but not in PMA-resistant or intermediate cells. Both Ras and Erk are robustly activated by PMA in all PMA-sensitive clones. In contrast, there is no detectable PMA-induced Ras activation in PMA-resistant clones, correlating with the defect in PMA-induced Erk activation. Interestingly, the intermediate clones activate Erk to a moderate extent, but do not express RasGRP and do not activate Ras in response to PMA. Thus, the results obtained for intermediate clones indicate that alternative pathways for PMA-induced Erk activation exist. Transfection of RasGRP enhances PMA-induced Erk activation in resistant cells, while the PMA response is inhibited by siRNA targeted at RasGRP in sensitive cells. We conclude that the differential effects of PMA on Erk activation in EL4 cell lines reflect differences in Erk activation pathways, and that RasGRP is one of the proteins responsible for PMA response. (Supported by NIH CA094144-01)

Protein Kinase C Delta Induces Apoptosis of Vascular Smooth Muscle Cells through Induction of the Tumor Suppressor p53 by Both p38-dependent and p38-independent Mechanisms

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. In the present study, we examined the novel PKC isoform protein kinase C delta (PKCdelta) and its role in vascular SMC apoptosis. In A10 SMCs, overexpression of PKCdelta was sufficient to induce apoptosis, whereas inhibition of PKCdelta diminished H2O2-induced apoptosis. Moreover, evidence is provided that the tumor suppressor p53 is an essential mediator of PKCdelta-induced apoptosis in SMCs. Activation of PKCdelta led to accumulation as well as phosphorylation of p53 in SMCs; this induction correlated with apoptosis. Furthermore, blocking p53 induction with small interference RNA or targeted gene deletion prevented PKCdelta-induced apoptosis, whereas restoring p53 expression rescued the ability of PKCdelta to induce apoptosis in p53 null SMCs. We also establish that PKCdelta regulates p53 at both transcriptional and post-translational levels. Specifically, the transcriptional regulation required p38 MAPK, whereas the post-translational modification, at least for serine 46, did not involve MAPK. Additionally, PKCdelta, p38 MAPK, and p53 co-associate in cells under conditions favoring apoptosis. Together, our data suggest that SMC apoptosis proceeds through a pathway that involves PKCdelta, the intermediary p38 MAPK, and the downstream target tumor suppressor p53.

Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate

The apical membrane Na(+)/H(+) exchanger isoforms NHE2 and NHE3 are involved in transepithelial Na(+) absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in the transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate NHE2 promoter expression in C2BBe1 cells. A small DNA region (-85/+249) was involved in the regulation of basal transcriptional activity of the NHE2 promoter as determined by transient transfection assays. RT-PCR analysis showed that NHE2 mRNA was upregulated in response to phorbol 12-myristate 13-acetate (PMA). Results from actinomycin D-treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in promoter activity through elements located on the -415/+249 DNA fragment. A PMA-induced nuclear factor that bound to the NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA response elements in the -415/+1 promoter region that bind to Sp1 and Sp3 in untreated nuclear extracts and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.

Role of α1 2.3 Subunit I-II Linker Sites in the Enhancement of Cav 2.3 Current by Phorbol 12-Myristate 13-Acetate and Acetyl-β-methylcholine

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.

Chronic oxidative stress reduces the respiratory burst response of neutrophils from beta‐thalassaemia patients

Beta-thalassaemia patients are susceptible to infections by mechanisms that are not fully understood. Polymorphonuclear neutrophils (PMN) destroy microbes by producing a burst of reactive oxygen species (ROS) (respiratory burst) in response to bacterial components, as well as to phorbol-myristate-acetate (PMA). In the present study, we compared ROS generation by normal and beta-thalassaemia PMN and assessed their response to PMA. Blood cells were subjected to gelatin separation, staining with dichlorofluorescin-diacetate and flow cytometry. At basal level, the fluorescence (mean fluorescence channel) of normal and thalassaemia PMN were 12.7 +/- 4.5 and 95.6 +/- 19.8 respectively; it changed to 283.4 +/- 72.5 and 39.5 +/- 14.3, respectively, upon PMA stimulation, indicating that thalassaemia PMN have a higher basal ROS but a reduced response to PMA. When normal PMN were treated with the oxidants hydrogen peroxide and butyl-hydroxyperoxide, as well as iron and haemin, which are elevated in thalassaemia, their basal ROS increased 5-22-fold, but the PMA response was abolished. Treating thalassaemic PMN with antioxidants (N-acetyl-L-cysteine or vitamins C and E) reduced their basal ROS but enhanced their PMA response. Our findings indicate that chronically stressed PMN, e.g. in thalassaemia, have reduced capacity to elicit a respiratory burst, which may compromise their antibacterial capacity, and imply prophylactic treatment with antioxidants for recurrent infections.

Chronic Oxidative Stress Reduces the Oxidative Burst Response of Thalassemic Polymorphonuclear Neutrophils.

Bacterial infections are a serious complication in the beta-thalassemia syndrome, but the mechanisms underlying the increased susceptibility to these infections are not fully understood. Factors, likely to be involved are anemia, splenectomy, iron-overload, alterations in immune responses and defective phagocytic cells. Polymorphonuclear neutrophils (PMN) destroy microbial organisms by a burst of reactive oxygen species (ROS) in response to bacterial components as well as to phorbol myristate acetate (PMA). The respiratory burst is mediated by a multi-component enzyme - NADPH oxidase. We previously showed that in thalassemia, RBC and platelets are under oxidative stress, which may affect their functionality and integrity, leading to premature hemolysis and platelet activation.In the present study, we utilized flow cytometric techniques to compare ROS generation in normal (n=18) and in beta-thalassemic (n=10) PMN and to assess respiratory burst in response to stimulation by PMA. Peripheral blood samples were mixed with 3% gelatin, and following 30 min incubation the upper layer, containing PMN, platelets and some RBC, was collected. ROS generation was measured by FACS analysis following staining of the cells with 2′-7′-dichlorofluorescein (DCF). PMN were “gated” according to granularity and size. The identity of the cells in the PMN gate was verified by immuno-staining for CD11b and CD15, specific PMN antigens. The intensity of the resultant ROS-mediated cell fluorescence was linearly related to the height (intensity) of the respiratory burst.The average mean fluorescence channel (MFC) of DCF-stained PMN of thalassemia patients and of normal donors at basal level was 39 and 10, respectively. Upon activation with 100 ng/ml PMA for 15 min. at 37°C, while the average MFC of the normal PMN increased by 36-fold, the thalassemic PMN increased by only two and a half fold.These results indicate high basal ROS generation by thalassemic PMN, but a reduced response to PMA. To determine whether this abnormal behavior of thalassemic PMN was due to oxidative stress, normal PMN were treated with various oxidants followed by DCF staining. Pre-treatment with hydrogen peroxide (1 mM), butyl-hydroxyperoxide (0.6 mM), hemin (0.1 mM) or ferric ammonium citrate (0.1 mM) increased the basal level of ROS generation (5 to 22-fold, but decreased the PMA response by 20–30%. In contrast, overnight treatment of thalassemic PMN with antioxidants such as N-acetyl-L-cysteine and vitamin C (both at 0.05 mM) reduced the basal ROS level and enhanced the PMN response to PMA.Our findings suggest that the oxidative status of PMN plays an important role in the defense mechanism against bacterial infections. PMN that are under chronic oxidative stress, such as in thalassemia (e.g., as a result of iron-overload) have a reduced capacity to elicit a respiratory burst. This may explain the increased susceptibility of thalassemic patients to bacterial infections. Consequently, these findings raise the possibility of using antioxidants in thalassemia and other conditions associated with recurrent bacterial infections.

Identification of Sites Responsible for Potentiation of Type 2.3 Calcium Currents by Acetyl-β-methylcholine

To address mechanisms for the differential sensitivity of voltage-gated Ca2+ channels (Cav) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M(1) receptors and different Cav alpha1 subunits, all with beta1B,alpha2/delta subunits. Acetyl-beta-methylcholine (MCh) decreased Cav 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Cav 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Cav 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the alpha1 subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the alpha1 2.3 subunits that respond to MCh. Quadruple Ser --> Ala mutation of the predicted MCh sites in the alpha1 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II-III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser --> Ala mutation of just the II-III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser --> Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II-III linker of alpha1 2.3 subunits mediate the responsiveness of Cav 2.3 channels to MCh. Because Cav 2.3 channels contribute to action potential-induced Ca2+ influx, these sites may account for M1 receptor-mediated regulation of neurotransmission at some synapses.

DNase I Hypersensitivity Patterns of the Serglycin Proteoglycan Gene in Resting and Phorbol 12-Myristate 13-Acetate-stimulated Human Erythroleukemia (HEL), CHRF 288-11, and HL-60 Cells Compared with Neutrophils and Human Umbilical Vein Endothelial Cells

We mapped the DNase I-hypersensitive sites (DHSS) of the serglycin gene in resting and phorbol 12-myristate 13-acetate (PMA)-stimulated human erythroleukemia (HEL) and CHRF 288-11 cells, which have megakaryocytic characteristics, and HL-60 promyelocytic leukemia cells. We compared these DHSS with those of normal primary neutrophils and human umbilical vein endothelial cells. Several DHSS appear to be involved in regulating the level of endogenous expression and in the PMA response of hematopoietic cell lines. A DHSS unique to resting HL-60 cells and induced in CHRF 288-11 by PMA may explain the high degree of endogenous expression in HL-60 relative to HEL and CHRF (Schick, B. P., Petrushina, I., Brodbeck, K. C., and Castronuevo, P. (2001) J. Biol. Chem. 276, 24726-24735). A total of 4 DHSS in intron 1 and 6 in intron 2 are associated with the PMA response in a cell-specific manner. A DHSS in the 5'-flanking region and another in intron 1 lie in areas that have high homology with the orthologous murine serglycin locus and are rich in potential transcription factor binding sites. One DHSS in intron 1 and one in intron 2 are located within Alu repeats. Two DHSS found in DNA of normal primary neutrophils were different from those of the cell lines. One DHSS in exon 2 unique to neutrophils correlated with a previously unrecognized alternative splicing that removes exon 2. Human umbilical vein endothelial cells had a DHSS in intron 1 that was common with the cell lines. The different patterns of DHSS exhibited by the cells studied suggest that cell- and differentiation-specific alterations in chromatin structure may control serglycin gene expression.

Low-molecular-weight heparin prevents high glucose- and phorbol ester-induced TGF-β;1 gene activation

Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression. Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays. The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site. LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.

Increased potential for high reactive oxygen species generation in pure sperm from leukocytospermic patients.

Objective: Phorbol 12-myristate 13-acetate (PMA) is the most powerful stimulant of cellular generation of reactive oxygen species (ROS). Enhanced PMA response has been correlated with poor semen quality, reduced sperm fertilizing potential and male infertility. The objective of this study was to investigate PMA response in pure sperm (free of leukocyte contamination) obtained from leukocytospermic patients. Design: A prospective clinical study in a male infertility clinic. Materials/Methods: Semen samples from 48 patients were examined according to the WHO criteria. Patients were classified into 2 groups, leukocytospermic (n = 16) and non-leukocytospermia (n = 32). Samples from normal donors (n = 13) were used as controls. Leukocytes were removed from the sperm suspensions using Dynabeads coated with anti CD45 antibodies and the lack of residual leukocyte contamination was confirmed by the formyl methionyl leucyl phenylalanine (FMLP) provocation test. Basal levels of ROS were determined in pure sperm suspensions and PMA provocation test was used to assess the residual capacity of sperm cells for ROS production. The results were expressed as ×106 counted photons per minute (cpm)/20 × 106 sperm/mL. Results: ROS levels [median (interquartile range 25% and 75%)] were significantly higher in the leukocytospermic group [3.2 (0.65, 6.8)] compared to the non-leukocytospermic group [0.31 (0.09, 1.2)] (P = 0.002), and donors [0.06 (0.01, 0.2)] (P = 0.001). Complete removal of leukocytes was confirmed by negative FMLP response. PMA-induced ROS generation was significantly higher in pure sperm from leukocytospermic samples [9.2 (2, 373.7)] compared to the non-leukocytospermic samples [0.94 (0.3, 2)] (P = 0.0003), and the donors [0.1 (0.03, 0.1)] (P = 0.0002). A significant positive correlation was observed between PMA-induced ROS in pure sperm and seminal leukocytic counts (r = 0.56, P < 0.0001), while PMA-induced ROS in pure sperm was negatively correlated with sperm concentration (r = 0.65, P < 0.0001), sperm motility (r = 0.44, P = 0.0003), and WHO morphology (r = 0.47, P = 0.0001). Conclusions: Our study demonstrates a strong correlation between leukocytospermia and enhanced sperm-derived PMA response. Increased residual capacity for ROS production in sperm from leukocytospermic samples reflects the poor quality of these sperm. Effective medical treatments to control leukocytospermia in these patients may result in improvement of their sperm quality. Supported by: The Cleveland Clinic Foundation.