Water Extract of Pleurotus eryngii var. ferulae Prevents High-Fat Diet-Induced Obesity by Inhibiting Pancreatic Lipase.

Pleurotus eryngii var. ferulae (PEF) is traditionally used in the prevention and treatment of lifestyle-related diseases. In this study, we investigated the ability of PEF extract to prevent obesity and metabolic diseases and explored the underlying mechanism. Mice were fed a high-fat diet (HFD) containing PEF extract for 12 weeks, and their body weight, adipose tissue and liver weights, and lipid profiles and blood glucose levels, were monitored. Fecal triglyceride (TG) levels were also measured and olive oil-loading tests were performed. Furthermore, the effect of PEF extract on pancreatic lipase (PL) activity was examined in vitro. Treatment with PEF extract for 12 weeks resulted in a significant decrease in the HFD-induced increases in body weight, white adipose tissue weight, liver weights, and lipid profiles, and improved glucose tolerance and insulin sensitivity. To assess the mechanism underlying the effect of PEF extract on obesity and diabetes, we investigated its role in inhibiting lipid absorption. Consumption of an HFD containing PEF extract significantly increased the TG level in feces compared with the controls, suggesting inhibition of TG absorption in the digestive tract. Furthermore, PEF extract suppressed the increase in serum TG levels resulting from oral administration of a lipid emulsion to mice, confirming inhibition of TG absorption. Moreover, PEF extract inhibited PL activity in vitro. Our combined results indicate that the anti-obesity and antidiabetic effect of PEF extract in mice fed an HFD may be caused by inhibition of lipid absorption as a result of reduced PL activity.

NOTUM Is Involved in the Progression of Colorectal Cancer.

BACKGROUND: There are limitations to current colorectal cancer (CRC)-specific diagnostic methods and therapies. Tumorigenesis proceeds because of interaction between cancer cells and various surrounding cells; discovering new molecular mediators through studies of the CRC secretome is a promising approach for the development of CRC diagnostics and therapies. MATERIALS AND METHODS: A comparative secretomic analysis was performed using primary and metastatic human isogenic CRC cells. Proliferation was determined by MTT and thymidine incorporation assay, migration was determined by wound-healing assay (ELISA). The level of palmitoleoyl-protein carboxylesterase (NOTUM) in plasma from patients with CRC was determined by enzyme-linked immunosorbent assay. RESULTS: NOTUM expression was increased in metastatic cells. Proliferation was suppressed by inhibiting expression of NOTUM. Knockdown of NOTUM genes inhibited proliferation as well as migration, with possible involvement of p38 and c-JUN N-terminal kinase in this process. The result was verified in patients with CRC. CONCLUSION: NOTUM may be a new candidate for diagnostics and therapy of CRC.

Reptin regulates pluripotency of embryonic stem cells and somatic cell reprogramming through Oct4-dependent mechanism.

Oct4 has been implicated in regulation of pluripotency in embryonic stem cells (ESCs) and reprogramming of somatic cells into induced pluripotent stem cells. However, the molecular mechanisms involved in Oct4-dependent regulation of pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Oct4-mediated self-renewal of ESCs and reprogramming of somatic cells, we attempted to identify Oct4-binding proteins using affinity purification and mass spectrometry. We identified Reptin, a key component of ATP-dependent chromatin remodeling complexes, as an Oct4-binding protein. Depletion of endogenous Reptin using lentiviral short hairpin RNA (shRNA) led to a decrease in the number and size of alkaline phosphatase-positive colonies of mouse ESCs. In addition, shRNA-mediated silencing of Reptin resulted in decreased expression of pluripotency-specific marker genes, including Oct4, Sox2, Nanog, and SSEA-1. Results of the Oct4 reporter assay showed synergism between Oct4 and Reptin, and depletion of endogenous Reptin abolished Oct4 transcriptional activity. Results of a chromatin immunoprecipitation assay showed the overlapping interaction of Reptin and Oct4 to CR4 in the Oct4 enhancer in ESCs. Knockdown of Reptin using shRNA suppressed the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, whereas overexpression of Reptin resulted in enhanced efficiency of induced pluripotent stem cell generation. These results strongly suggest that Reptin plays a key role in maintaining the pluripotency of ESCs and in establishing the pluripotency during reprogramming of somatic cells by regulation of Oct4-mediated gene regulation.

Xanthene derivatives increase glucose utilization through activation of LKB1-dependent AMP-activated protein kinase.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

Proteomic analysis of hypoxia-induced U373MG glioma secretome reveals novel hypoxia-dependent migration factors.

High-grade gliomas are one of the most common brain tumors and notorious for poor prognosis due to their malignant nature. Gliomas have an extensive area of hypoxia, which is critical for glioma progression by inducing aggressiveness and activating the angiogenesis process in the tumor microenvironment. To resolve the factors responsible for the highly malignant nature of gliomas, we comprehensively profiled the U373MG glioma cell secretome-exosome and soluble fraction under hypoxic and normoxic conditions. A total of 239 proteins were identified from the exosome and soluble fractions. Vascular endothelial growth factor, stanniocalcin 1 (STC1) and stanniocalcin 2, and insulin-like growth factor binding protein 3 and 6, enriched in the soluble fraction, and lysyl oxidase homolog 2 enriched in the exosomal fraction were identified as upregulated proteins by hypoxia based on a label-free quantitative analysis. STCs and insulin-like growth factor binding proteins, which were identified as secretory proteins under hypoxic conditions, were highly correlated with glioma grade in human patients by microarray analysis. An in vitro scratch wound assay revealed that STC1 and 2 have important functions in the induction of cell migration in a hypoxia-dependent manner, suggesting that they are hypoxia-dependent migration factors.

Comparative secretome analysis of human bone marrow-derived mesenchymal stem cells during osteogenesis.

Osteogenesis is a tightly regulated process that involves coordinated extracellular signals from autocrine and paracrine loops. Secretory proteins during osteogenesis can inhibit cell proliferation and activate cell differentiation toward mature osteoblasts, which are characterized by mineralization. In this study, we attempted to identify these secretory proteins during osteogenesis using LC-MS/MS analysis. We compared the secretome between undifferentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) and differentiated osteoblasts. Among 315 proteins that were identified, 177 proteins were present at increased levels in osteoblasts, whereas 88 proteins were present at decreased levels. Among the identified proteins, several were validated by quantitative RT-PCR and immunoblot analysis. Of particular interest, calcium homeostasis-related proteins were upregulated, whereas stem cell proliferation-related proteins and other lineage-related proteins were downregulated during osteogenesis. These findings provide information about the dynamic changes in the expression and secretion of proteins during osteogenesis and suggest the putative role of secretory proteins in osteogenesis.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the ß1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Proteomic identification of ADAM12 as a regulator for TGF-ß1-induced differentiation of human mesenchymal stem cells to smooth muscle cells.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-ß1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs. METHODS AND RESULTS: Pretreatment of hASCs with the lipid raft disruptor methyl-ß-cyclodextrin abrogated TGF-ß1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-ß1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-ß1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-ß1 treatment. TGF-ß1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-ß1-induced differentiation of hASCs into smooth muscle cells. CONCLUSIONS: These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-ß1-induced differentiation of hASCs into smooth muscle cells.

Secretomics for skeletal muscle cells: a discovery of novel regulators?

Metabolic tissues, including skeletal muscle, adipose tissue and the digestive system, dynamically secrete various factors depending on the metabolic state, communicate with each other and orchestrate functions to maintain body homeostasis. Skeletal muscle secretes cytokines such as interleukin-6 (IL-6), IL-15, fibroblast growth factor-21 (FGF21) and IL-8. These compounds, myokines, play important roles in biological homeostasis such as energy metabolism, angiogenesis and myogenesis. New technological advances have allowed secretomics - analysis of the secretome - to be performed. The application of highly sensitive mass spectrometry makes qualitative and quantitative analysis of the secretome of skeletal muscle possible. Secretory proteins derived from skeletal muscle cells under various conditions were analyzed, and many important factors were suggested. In-depth studies of the secretome from metabolic cells in various conditions are strongly recommended. This study will provide information on methods of novel communication between metabolic tissues.

Proteomic identification of betaig-h3 as a lysophosphatidic acid-induced secreted protein of human mesenchymal stem cells: paracrine activation of A549 lung adenocarcinoma cells by betaig-h3.

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified ßig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced ßig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced ßig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced ßig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of ßig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of ßig-h3 in hASCs potentiated it. Furthermore, recombinant ßig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived ßig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Proteomic analysis of tumor necrosis factor-alpha-induced secretome of human adipose tissue-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to tumor necrosis factor-alpha (TNF-alpha) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-alpha treatment. The TNF-alpha-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-alpha also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-alpha-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL-8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNF-alpha-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis.

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label-free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT-PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor-beta (TGF-beta) signaling changed during adipogenesis. The expressions of secreted frizzled-related proteins, dickkopf-related proteins, and latent TGF-beta-binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF-beta signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity-related metabolic diseases.

Lysophosphatidylcholine activates adipocyte glucose uptake and lowers blood glucose levels in murine models of diabetes.

Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cdelta inhibitor) and by the adenoviral expression of dominant negative protein kinase Cdelta. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.

Novel compound 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) prevents 2,3,7,8-TCDD-induced toxicity by antagonizing the aryl hydrocarbon receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant with many toxic effects, including endocrine disruption, reproductive dysfunction, immunotoxicity, liver damage, and cancer. These are mediated by TCDD binding to and activating the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor. In this regard, targeting the AhR using novel small molecule inhibitors is an attractive strategy for the development of potential preventive agents. In this study, by screening a chemical library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), that potently inhibits TCDD-induced AhR-dependent transcription. In addition, CH-223191 blocked the binding of TCDD to AhR and inhibited TCDD-mediated nuclear translocation and DNA binding of AhR. These inhibitory effects of CH-223191 prevented the expression of cytochrome P450 enzymes, target genes of the AhR. Unlike many known antagonists of AhR, CH-223191 did not have detectable AhR agonist-like activity or estrogenic potency, suggesting that CH-223191 is a specific antagonist of AhR. It is noteworthy that CH-223191 potently prevented TCDD-elicited cytochrome P450 induction, liver toxicity, and wasting syndrome in mice. Taken together, these results demonstrate that this novel compound, CH-223191, may be a useful agent for the study of AhR-mediated signal transduction and the prevention of TCDD-associated pathology.

Ligand profiling and identification technology for searching bioactive ligands.

We introduce a new methodology named ligand profiling and identification for effective discovery of bioactive ligands such as peptide hormones. This technology was developed from a new concept of parallel column chromatography and active fraction profiling by nano-LC MS. Traditional methods use sequential column chromatography, and thus are inevitably limited by the low abundance of the peptide of interest and by a low yield due to the many column steps. Using this new technology, insulin was successfully identified and diarginylinsulin, a minor intermediate form of insulin, was unexpectedly also identified simultaneously from 100 mg of porcine pancreatic tissue. This integrative technology could be used to search for various low-abundance peptides (or bioactive molecules) rapidly and simultaneously, by applying this to the later stages of traditional sequential purification.

Leumorphin has an anti-apoptotic effect by activating epidermal growth factor receptor kinase in rat pheochromocytoma PC12 cells.

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, beta-neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor-binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition-binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src-dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation-induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, which occur independent of the kappa opioid receptor.

The novel phospholipase C activator, m-3M3FBS, induces monocytic leukemia cell apoptosis.

We investigated the effect of the novel phospholipase C activator, m-3M3FBS, on the apoptosis of leukemic cells. m-3M3FBS inhibited the growth of the leukemic cell lines U937 and THP-1, but not primary monocytes. m-3M3FBS induced the apoptosis of U937 cells, which was accompanied by chromatin condensation and DNA fragmentation. Moreover, m-3M3FBS-induced apoptosis appeared to involve the down-regulation of anti-apoptotic Bcl-2, the up-regulation of pro-apoptotic Bax, the release of cytochrome c, and caspase activation. m-3M3FBS-induced apoptosis of U937 cells was also partly inhibited by BAPTA-AM and EGTA, indicating the involvement of intracellular calcium signaling on the apoptosis in U937 cells. The results of our study suggest that m-3M3FBS can be developed as a novel anti-leukemic agent.

G2 arrest and apoptosis by 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS), a novel cytotoxic compound.

We screened a library of 11,000 small molecular weight chemicals, looking for compounds that affect cell viability. We have identified 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS) as a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy.

Differential activation of formyl peptide receptor-like 1 by peptide ligands.

Formyl peptide receptor-like 1 (FPRL1) plays a key role in the regulation of immune responses. The activation of FPRL1 induces a complicated pattern of cellular signaling, which results in the regulation of several immune responses, such as chemotactic migration and the production of reactive oxygen species (ROS). Because some of these cellular responses are not beneficial to the host, ligands that selectively modulate these cellular responses are useful. His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that binds to FPRL1. In this study, we generated various HFYLPM analogues and examined their effects on cellular responses via FPRL1 in FPRL1-expressing rat basophilic leukemia-2H3 cells or in primary human neutrophils. Among the HXYLPM analogues, His-Arg-Tyr-Leu-Pro-Met (HRYLPM) activated a broad spectrum of cellular signaling events, including an intracellular Ca(2+) concentration increase, phosphoinositide 3-kinase, extracellular signal-regulated kinase, and Akt activation, however, His-Glu-Tyr-Leu-Pro-Met (HEYLPM) activated only intracellular Ca(2+) concentration and Akt but did not increase Ca(2+). In addition, HRYLPM was found to stimulate chemotaxis and ROS generation via phosphoinositide 3-kinase and an intracellular Ca(2+) concentration increase, respectively, whereas HEYLPM stimulated chemotaxis but not ROS generation. With respect to the molecular mechanisms involved in the differential action of HRYLPM and HEYLPM, we found that HRYLPM but not HEYLPM competitively inhibited the binding of (125)I-labeled Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm, a FPRL1 ligand) to FPRL1. This study demonstrates that the important chemoattractant receptor, FPRL1, may be differentially modulated by distinct peptide ligands. We also suggest that HRYLPM and HEYLPM may be used to selectively modulate FPRL1.