Involvement of ERK-1/2 in IL-21-induced cytokine production in leukemia cells and human monocytes.

Cytokines play an important role in the immune system, and abnormalities in their production have been found in many human diseases. Interleukin-21 (IL-21), a type I cytokine produced by activated T cells, has diverse effects on the immune system, but its ability to induce production of other cytokines is not well delineated. Furthermore, the signaling pathway underlying its action is poorly understood. Here, we have evaluated IL-21-induced cytokine production in human monocytes and U937 leukemia cells. We found that IL-21 induces upregulation of a variety of cytokines from multiple cytokine families. We also found that IL-21 triggers rapid activation of ERK1/2. Neutralizing antibody to the IL-21R prevented both IL-21-induced cytokine production and IL-21-induced activation of ERK1/2. Inhibition of ERK1/2 activity by the ERK-selective inhibitor U0126 reverses the ability of IL-21 to upregulate cytokine production, suggesting that IL-21-induced cytokine production is dependent on ERK1/2 activation.

SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer.

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation.

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.

Structural and functional comparison of a 22 kDa protein from internal human platelet membranes with cardiac phospholamban.

We have shown that a platelet 22 kDa phosphoprotein is similar to the cardiac regulator phospholamban, in that both are phosphorylated by cAMP- and calmodulin-dependent protein kinases, and that both can be phosphorylated simultaneously by these two classes of protein kinases to yield an additive stimulation of the respective Ca2+ pumps (Adunyah, S.E. and Dean, W.L. (1987) Biochim. Biophys. Acta 930, 401-409). However, whereas phosphorylation of phospholamban increases the affinity of the cardiac Ca2+-ATPase for Ca2+, phosphorylation of the platelet 22 kDa protein increased the Vmax of the pump. In addition, antibodies raised against canine phospholamban did not crossreact with the human platelet 22 kDa protein. Finally, it was not possible to dissociate the platelet protein into lower-molecular-weight subunits by boiling in sodium dodecylsulfate which is characteristic of cardiac phospholamban. These results show that although phosphorylation of low-molecular-weight membrane-associated regulator proteins in cardiac muscle and platelets appears to stimulate the respective Ca2+ pumps, these proteins have different chemical and physical properties.

Regulation of c-jun mRNA expression by hydroxyurea in human K562 cells during erythroid differentiation.

Hydroxyurea (HU) is an antitumor agent which also induces hemoglobinization during erythroid differentiation. In addition, HU stimulates the synthesis of fetal hemoglobin in sickle cell anemia patients. To further understand its mechanism of action, we investigated the effects of HU on regulation of c-jun expression prior to the onset of erythroid differentiation of K562 cells. HU induced a dose-dependent stimulation of c-jun synthesis. The levels of c-jun mRNA was elevated 4 to 7.5-fold by HU within 2 h. This was followed by a gradual decline to the basal level by 24 h. Both nuclear run-on and actinomycin D pulse experiments strongly indicate that HU regulates c-jun mRNA expression by increasing the rate of synthesis as well as stabilizing the c-jun mRNA. In addition, the level of jun protein was elevated by 2 to 5-fold within 4 h in HU treated cells. Furthermore, concentrations of HU below 250 microM slightly increased the 5X AP-1/CAT activity. These results strongly suggest that HU induces both transcriptional and post-transcription regulation of c-jun during erythroid differentiation.

Effect of phorbol esters and diacylglycerols on calcium transport by human platelet membranes.

The effects of phorbol esters and diacylglycerols on Ca2+ transport in isolated human platelet membranes were determined. Phorbol 12-myristate 13-acetate (PMA) stimulated Ca2+-ATPase activity in crude and purified internal platelet membranes approximately 2-fold with half-maximal stimulation occurring at 10 nM. Dilauroylglycerol also stimulated Ca2+-ATPase activity half-maximally at a concentration of 7.5 microM, but dioctanoylglycerol was without effect at up to 30 microM. PMA also inhibited Ca2+ uptake when added before or after commencement of ATP-dependent transport. PMA (25 nM) doubled the rate of Ca2+ efflux from passively loaded membranes in the absence of ATP. No protein kinase C activity was detected in crude or purified membranes by histone phosphorylation or endogenous protein phosphorylation assays. These results suggest that PMA and dilauroylglycerol stimulate Ca2+-ATPase activity and inhibit ATP-dependent Ca2+ transport by increasing the permeability of the membranes to Ca2+.

Subsequent Adenomas of Ileal Pouch and Anorectal Segment after Prophylactic Surgery for Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is an autosomally dominant disease characterized by the early development of colorectal adenomas and carcinoma in untreated patients. Patients with FAP may develop rectal cancer at their initial presentation (primary) or after prophylactic surgery (secondary). Controversies exist regarding which surgical procedure represents the best first-line treatment. The options for FAP are ileorectal anastomosis (IRA) or a restorative proctocolectomy (RPC) with either a handsewn or a stapled ileal pouch-anal anastomosis (IPAA), with or without mucosectomy. The purpose of these surgeries is to stop progression to an adenoma-cancer sequence by eradicating the colon, a disease prone organ. Unfortunately, these surgical procedures, which excise the entire colon and rectum while maintaining transanal fecal continence, do not guarantee that patients still won't develop adenomas. Based on the available literature, we therefore reviewed reported incidences of pouch-related adenomas that occurred post prophylactic surgery for FAP. The review consists of a collection of case, descriptive, prospective and retrospective reports. OBJECTIVES: To provide available data on the natural history of subsequent adenomas after prophylactic surgery (by type) for FAP. METHODS: A review was conducted of existing case, descriptive, prospective and retrospective reports for patients undergoing prophylactic surgery for FAP (1975 - August, 2013). In each case, the adenomas were clearly diagnosed in one of the following: the ileal pouch mucosa (above the ileorectal anastomosis), within the anorectal segment (ARS) below the ileorectal anastomosis, or in the afferent ileal loop. RESULTS: A total of 515 (36%) patients with pouch-related adenomas have been reported. Two hundred and eleven (211) patients had adenomas in the ileal pouch mucosa, 295 had them in the ARS and in 9 were in the afferent ileal loop. Patients with pouch adenomas without dysplasia or cancer were either endoscopically polypectomized or were treated with a coagulation modality using either a Nd:Yag laser or argon plasma coagulation (as indicated). Patients with dysplastic pouch adenomas or pouch adenomas with cancer had their pouch excised (pouchectomy). CONCLUSION: In patients with FAP treated with IRA or RPC with IPAA, the formation of adenomas in the pouch-body mucosa or ARS/anastomosis and in the afferent ileal loop is apparent. Because of risks for adenoma recurrence, a life time endoscopic pouch-surveillance is warranted.

Granulocyte-macrophage colony stimulating factor stimulates rapid phosphorylation of proteins on tyrosines in human U937 and HL-60 leukemic cells.

GM-CSF is a colony stimulating factor which regulates growth and differentiation of hematopoietic progenitors. Also, it stimulates increases in c-jun expression and activates AP-1 enhancer activity. However, it is not clearly known how its signal is transduced. In this study, the effect of GM-CSF on tyrosyl phosphorylation was examined in Human U937 and HL-60 Leukemic cells by Western Blotting using anti-phosphotyrosine monoclonal antibody and autoradiography. Evidence is presented that GM-CSF stimulates a rapid phosphorylation on tyrosines of several proteins of relative Mr. 150, 110, 95, 55, 48, and 15 kDa within 60 seconds. The effect of GM-CSF was blocked by the tyrosine kinase inhibitor ST638 but not by H-7. Both G-CSF and M-CSF were also effective in stimulating tyrosine phosphorylation. These results strongly support a role for tyrosine kinases in regulation of cellular events by GM-CSF in monoblasts.

Interleukin-11 induces rapid PKC activation and cytosolic to particulate translocation of alpha and beta PKC isoforms in human erythroleukemia K562 cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine which regulates the growth of hematopoietic progenitor cells and activates platelet maturation. Previous studies have shown that, IL-11 activates a set of signaling cascades involving the JAK/STAT and Raf/MAPK pathways. The purpose of the current studies was to obtain evidence about the possible involvement of PKC in the IL-11 signaling pathway. Evidence presented in this report suggests that IL-11 stimulates rapid PKC activation and markedly induces cytosolic to particulate (membrane) association of alpha and beta PKC isoforms. These findings provide preliminary evidence that PKC may be involved in the IL-11 signaling cascade.

Ca2+ transport in human platelet membranes. Kinetics of active transport and passive release.

Active Ca2+ transport and passive release were characterized in crude and purified human platelet membranes to facilitate comparison with skeletal muscle sarcoplasmic reticulum. Endoplasmic reticulum markers were enriched from 3- to 14-fold in the purified membranes, while surface membrane antigens were reduced 4-fold and mitochondrial contamination was completely eliminated. The pH optimum for active Ca2+ transport in platelet membranes was 7.6, and the optimum for Ca2+-ATPase activity ranged from 7.6 to 8.0. Upon addition of MgATP there was a burst in active Ca2+ transport activity. In the absence of phosphate, steady state was reached within 20 s; added phosphate promoted continued uptake for greater than 1 h. The maximum pump stoichiometry was 2.0 Ca2+/ATP. The Ca2+ ionophore A23187 caused rapid release of 90% of the sequestered Ca2+ in the presence of phosphate. The dependence of Ca2+ transport on MgATP was biphasic with apparent Km values of 0.6 mM and 9.5 microM. Kinetic measurements with varied external Ca2+ yielded a single Km of 0.1 microM. Mg2+ stimulated Ca2+ transport and Ca2+-ATPase activities. Results with crude and purified membranes were similar, and comparison with the Ca2+ pump from sarcoplasmic reticulum revealed nearly identical enzymatic properties. In contrast to the results of comparing active Ca2+ transport, the characteristics of Ca2+ release from platelet membranes were quite different from those of sarcoplasmic reticulum. External Ca2+ did not promote release of sequestered Ca2+ from platelet membranes in contrast to sarcoplasmic reticulum. In addition, spontaneous release of Ca2+ from platelet membranes did not occur after ATP depletion. Inositol trisphosphate induced rapid partial release of Ca2+ from platelet membranes but had no effect on sarcoplasmic reticulum under identical conditions. Thus active Ca2+ transport is quite similar in internal membranes of platelet and skeletal muscle, but the mechanism of Ca2+ release appears to be entirely different.

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes.

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.

Sodium butyrate stimulates PKC activation and induces differential expression of certain PKC isoforms during erythroid differentiation.

Sodium butyrate (NaB) is an differentiation inducer currently under clinical investigation as a potential therapy for the treatment of sickle cell disease and prostate cancer. Though the biologic effects of this agent is well documented, its mechanism of action remains largely known. The mechanisms by which it transduces its signal to the nucleus is the subject of intense investigation in our laboratory. In this report, we demonstrate that NaB stimulates PKC activation by 3-fold and induces differential expression of several PKC isoforms. Notably, it upregulates PKC epsilon and downregulates PKC beta during erythroid differentiation. These findings suggest that certain PKC isoforms may play important roles in the signal transduction mechanisms of this agent leading to regulation of erythroid proliferation and differentiation.

Sodium butyrate induces tyrosine phosphorylation and activation of MAP kinase (ERK-1) in human K562 cells.

Butyrate is a naturally occurring 4-carbon fatty acid. Biologically, butyrate has been shown to affect the morphology and growth rate of mammalian cells, as well as induce gene expression. Moreover, butyrate has been proven to serve as an anticancer agent, which unlike others (methotrexate and hydroxyurea), is a nontoxic, safe alternative to cancer treatment. It also induces erythroid differentiation in K562 cells. However, its mechanism of action has yet to be determined. In this study we investigated the effects of sodium butyrate (NaB) on tyrosine phosphorylation in K562 erythroleukemic cells. We demonstrate that NaB induces both dose and time-dependent tyrosine phosphorylation of several proteins, the effects of which were blocked by the tyrosine kinase inhibitor genistein. Furthermore, NaB induces tyrosine phosphorylation and rapid activation of MAP kinase (ERK-1). These findings provide the first evidence that the signal transduction mechanism of NaB involves rapid tyrosine phosphorylation and activation of MAP kinase.

Inositol triphosphate-induced Ca2+ release from human platelet membranes.

Inositol (1,4,5) triphosphate (IP3) was observed to induce release of sequestered Ca2+ from crude human platelet membranes. This activity was also shown to be present in purified membranes enriched in Ca2+-ATPase activity. Maximal Ca2+ release occurred at 8 microM IP3 and half maximal activity was at 0.4 microM. Release was quite rapid and was complete by 40 s. Released Ca2+ was pumped back into the membrane vesicles and the rate of this reuptake was increased by the presence of phosphate. These results demonstrate that internal platelet membranes that possess an active Ca2+-pump will release sequestered Ca2+ in the presence of the second messenger IP3. IP3 did not induce release of Ca2+ from skeletal muscle sarcoplasmic reticulum when ATP was present.

Evidence for the involvement of JAK/STAT pathway in the signaling mechanism of interleukin-17.

Interleukin-17 is a T-cell-derived pro-inflammatory cytokine, exhibiting multiple biological activities in a variety of cells and believed to fine tune all general phases of hematopoietic response. However, the signaling mechanism of this novel cytokine remains unknown. Here, we report for the first time that the early signaling events triggered by interleukin-17 involve tyrosine phosphorylation of several members of the JAK and STAT proteins in human U937 monocytic leukemia cells. Immunoprecipitation with specific antibodies followed by Western blot analysis with antiphosphotyrosine antibody has shown that in U937 cells, interleukin-17 induces time-dependent stimulation of tyrosine phosphorylation of JAK 1, 2 and 3, Tyk 2 and STAT 1, 2, 3 and 4 within 0.5 to 30 min. Interleukin-17-mediated tyrosine phosphorylation of these proteins strongly suggests that the JAK/STAT signaling pathway may play a major role in transducing signals from interleukin-17 receptors to the nucleus.

Evidence for the involvement of LCK and MAP kinase (ERK-1) in the signal transduction mechanism of interleukin-15.

Human IL-15 is a cytokine expressed by a variety of tissues and cells including myeloid progenitor cells and monocytes. It shares biologic properties of IL-2 and utilizes the beta subunit of the IL-2R. IL-15 regulates proliferation of activated B and NK cells and stimulates chemoattraction in blood T-lymphocytes, effects which are inhibited by an anti-IL-2R beta antibody. Because little is known about the mechanism(s) by which IL-15 signal is transduced, this study was conducted to identify some of the key molecules involved in IL-15-induced signaling cascade(s). We report that IL-15 induces tyr phosphorylation of the p75IL-2R beta and p64IL-2R gamma subunits and Shc. Also, it activates both p56lck and MAPK (ERK-1). These results strongly suggest that LCK and MAPK may play vital roles in mediation of cellular activation by IL-15.

Calcium uptake and inositol trisphosphate-induced calcium release from parathyroid gland membranes.

The manner in which changes in Ca2+ are translated by the parathyroid gland into a signal for secretion of its major secretory proteins, parathormone and secretory protein-I, is not understood. In order to gain insight into this mechanism, Ca2+ transport was evaluated in porcine parathyroid gland membranes prepared by homogenization and differential centrifugation. The membranes were considered to be intact and appropriately 'sided' since they contained secretory protein-I and exhibited ATP-dependent uptake of [45Ca2+]. About 1 mol of ATP was hydrolyzed per mol of Ca2+ transported, signifying that the ATPase activity was relevant to the Ca2+ transport system of the membranes. Inositol trisphosphate, which is believed to be a universal signal for generating a change in cytosolic Ca2+ by discharging Ca2+ from internal membranes, caused the parathyroid membranes to release accumulated Ca2+. The membranes were able to partially reaccumulate the discharged Ca2+. The data suggest that intracellular Ca2+ metabolism in the parathyroid cell is similar to that of other secretory cells. If a change in intracellular concentration of this ion is involved in secretion, it may occur locally within the cytosol.

Interleukin-13 induces rapid tyrosine phosphorylation and activation of Raf-1 kinase in human monocytic progenitor cell line U937.

IL-13 is a pleiotropic cytokine produced by Th0, Th1-like, and CD8 T cells in response to antigen stimulation. Its biological effects include suppression of cytotoxic activity of monocytes/macrophages and suppression of pro-inflammatory cytokine production. However, the mechanism of IL-13 remains unknown. In this study we investigated the effects of rhIL-13 on tyrosine phosphorylation in U937 monocytic progenitor cells by immunoblotting and immunocomplex kinase assays. We demonstrate that rhIL-13 stimulates dose-dependent tyrosine phosphorylation of several proteins of Mr. 93, 80, 74, 49.5, 42, 30, 22 and 18 kDa within 30 sec. The effect of IL-13 was blocked by the tyrosine kinase inhibitor erbstatin. Furthermore, IL-13 induces tyrosine phosphorylation and rapid activation of raf-1 kinase. These findings provide the first evidence that the mechanism of IL-13 involves rapid tryrosine phosphorylation and activation of raf-1 serine/threonine kinase.

Regulation of jun expression and activation of AP-1 activity by erythropoietin.

Erythropoietin (EPO) is a molecule which regulates hemoglobin gene expression during erythroid proliferation and differentiation. However, the mechanism by which it regulates gene expression is poorly understood. Secondly, unlike other hematopoietic factors which induce expression of early response genes, it is unknown whether EPO is capable of inducing the expression and activation of early response genes. In this study, evidence is presented that EPO induces the expression of early response genes c-jun, junD and c-fos mRNA, stimulates jun protein synthesis and induces activation of 5 x AP-1/CAT activity in hyman erythroleukemia K562 cells. Also, EPO appears to regulate jun expression at transcriptional, post transcriptional and translational levels. These observations suggest that jun and fos expression may be relevant in the mechanism of growth control by EPO.