Gene expression profiling during early folliculogenesis in the mouse ovary.

OBJECTIVE: To evaluate genes involved in ovarian primordial-to-primary follicle transition. DESIGN: Experimental animal study. SETTING: Research institute. ANIMAL(S): Day-2 and day-4 female Swiss mice. INTERVENTION(S): We conducted a complementary DNA array study using ovarian messenger RNAs from day-2 and day-4 mice. MAIN OUTCOME MEASURE(S): The expression profiles of 1,176 genes in neonatal mouse ovaries on day 2 and day 4, which contain primordial and primary follicles, respectively, were compared. RESULT(S): Twenty-six percent of genes were differentially expressed between day-2 and day-4 ovaries, with 19% being up-regulated and 7% down-regulated in day 4. Analysis of differentially expressed genes revealed that the primordial-to-primary follicle stage transition is associated with induction in the expression of mainly growth factors, immune-related factors, hormone and hormone receptors, and signal transducers. The transition is also associated with proliferation of granulosa cells and absence of apoptosis. In addition, our studies demonstrated that the primary follicles express estrogen receptor beta and are responsive to estrogen actions in vitro in terms of increase in the number of primary follicles and granulosa cell proliferation. CONCLUSION(S): The transition of primordial to primary follicles is associated with the participation of multiple pathways in regulating gene expression.

Interferon-beta reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Effect of genistein on the expression of bone metabolism genes in ovariectomized mice using a cDNA microarray.

Osteoporosis associated with estrogen deficiency is defined as an abnormal decrease in bone mass leading to an increased fracture risk. Genistein (GEN), as a phytoestrogen, is a type of soybean-derived isoflavone that possesses structural similarity to estrogen. In this study, we assessed the effect of GEN in ovariectomized (OVX) mice. To determine the effect of GEN on bone metabolism, we investigated gene expression profiles using a radioactive cDNA microarray. Eight-week-old female mice were either sham operated (SHAM) or OVX. From 1 week after the operation, OVX mice were injected daily with intraperitoneal GEN (0.1, 0.5, 1.5 and 3.0 mg/day) or 17beta-estradiol (E2, 0.03 microg/day) for 4 weeks. A cDNA microarray was used to evaluate changes in the expression of 1,152 genes. OVX mice showed bone mineral density (BMD) loss versus SHAM mice (5.8+/-0.4 vs. 6.9+/-0.6 mg/cm2). However, femur BMDs were completely restored by GEN and by E2 administration in OVX mice. Serum osteocalcin in OVX mice treated with 0.5 mg/day of GEN was 1.6-fold (44.30+/-5.73 ng/ml) higher than that in untreated mice. GEN treatment up-regulated 38 genes (e.g., mitogen-activated protein kinase 10) and down-regulated 18 (e.g., matrix metalloproteinase 13). Moreover, GEN was found to have a protective effect on bone loss caused by estrogen deficiency in OVX mice. The present study suggests that GEN modulates bone metabolism-related gene expression, including calciotropic receptor, cytokines, growth factors and bone matrix proteins.

A study of Q-switched Nd:YAG laser irradiation and paracrine function in human skin cells.

BACKGROUND AND OBJECTIVES: This preliminary laboratory-based study looks at the paracrine release from human skin cells subject to sublethal Q-switched Nd:YAG 532 nm laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Human dermal fibroblast and keratinocyte cultures were exposed to sublethal energy using the Nd:YAG 532 nm laser. Altered gene expression was then screened using RT-PCR for a range of paracrine factors known to affect melanogenesis, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), stem cell factor (SCF), melanocyte stimulating hormone (MSH), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and protease-activated receptor-2 (PAR-2). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein production. Conditioned medium was used to assess altered melanogenesis in a melanoma cell line. RESULTS: Fibroblasts exposed to sublethal radiation showed upregulation of b-FGF, HGF and SCF. This contrasts with keratinocytes which showed upregulation of IL-6. Elevated protein levels of b-FGF and SCF were confirmed by ELISA assay. Conditioned fibroblast medium was shown to stimulate melanogenesis in a melanoma cell line. CONCLUSIONS: This preliminary laboratory study reports, for the first time, specific gene upregulation using the Q-switched Nd:YAG 532 nm laser.

Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period.

To continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14(+) cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3-7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants.

Effects of colostrum feeding and dexamethasone treatment on mRNA levels of insulin-like growth factors (IGF)-I and -II, IGF binding proteins-2 and -3, and on receptors for growth hormone, IGF-I, IGF-II, and insulin in the gastrointestinal tract of neonatal calves.

The somatotropic axis regulates growth of the gastrointestinal tract (GIT). In addition, colostrum feeding and glucocorticoids affect maturation of the GIT around birth in mammals. We have measured mRNA levels of members of the somatotropic axis to test the hypothesis that colostrum intake and dexamethasone treatment affect respective gene expression in the GIT. Calves were fed either colostrum or an isoenergetic milk-based formula, and in each feeding group, half of the calves were treated with dexamethasone (DEXA; 30 microg/kg body weight per day). Individual parameters of the somatotropic axis differed (P < 0.05) among different GIT sections and formula feeding increased (P < 0.05) mRNA levels of individual parameters at various sites of the GIT. Effects of DEXA on the somatotropic axis in the GIT partly depended on different feeding. In colostrum-fed calves, DEXA decreased (P < 0.05) mRNA levels of IGF-I (esophagus, fundus, duodenum, and ileum), IGF-II (fundus), IGFBP-2 (fundus), IGFBP-3 (fundus), IGF1R (esophagus, ileum, and colon), IGF2R (fundus), GHR (fundus), and InsR (esophagus, fundus), but in formula-fed calves DEXA increased mRNA levels of IGF-I (esophagus, rumen, jejunum, and colon). Furthermore, DEXA increased (P < 0.05) mRNA levels of IGF-II (pylorus), IGFBP-3 (duodenum), IGF2R (pylorus), and GHR (ileum), but decreased mRNA levels of IGFBP-2 (ileum), and IGF1R (fundus). Whereas formula feeding had stimulating effects, effects of DEXA treatment on the gene expression of parameters of the somatotropic axis varied among GIT sites and partly depended on feeding.

The effect of ethanol exposure on extraembryonic vascular development in the chick area vasculosa.

The effect of ethanol (EtOH) exposure on extraembryonic vascular development was examined using the chick embryo area vasculosa (AV) in shell-less culture. Embryos were placed in cultures at Hamburger Hamilton (HH) stage 11/12 and a single dose of EtOH (10, 30 or 50%) was applied to the center of the blastodisc. Untreated/sodium-chloride-treated controls showed normal embryonic growth and well-developed extraembryonic vessels at 24/48 h of treatment. At doses of 30 and 50%, the mortality rate was significantly increased, and survivors demonstrated significant growth retardation and inhibition of normal vascular development in a dose-dependent manner. Immunostaining for vascular endothelial growth factor (VEGF) showed that mesenchymal cells continued to differentiate into angioblasts to form blood islands, but their assembly into primitive vessels was perturbed in a dose-dependent manner. Northern blot analyses of basic fibroblast growth factor, VEGF, Flt-1 and Flk-1 mRNA expression supported these findings and showed a dose-dependent decrease in EtOH-treated cultures compared to controls. Co-treatment with alpha-tocopherol (0.05 M) or all-trans-retinoic acid (10(-8) M) significantly decreased the mortality rate and improved both embryonic growth and extraembryonic vascular development in the cultures. On the other hand, almost all embryos treated with 10% EtOH survived the first 48 h after treatment. However, the complexity of the vascular tree measured as the relative vasculogenesis index, the surface area of the AV and the mRNA expression of vasculogenic molecules were increased during the first 24 h. This acute effect disappeared 48 h after treatment and the vascular tree continued to develop parallel to the controls. No significant growth retardation was observed in this group. These results suggest that, in terms of extraembryonic vascular development, an early, single, low-dose EtOH exposure may have an acute, short-term positive effect, whereas moderate- or high-dose EtOH exposure may severely perturb this process disabling the necessary absorption of the nutrients for the embryo to develop properly. The mechanisms of action of EtOH on extraembryonic vascular development may involve the establishment of reactive oxygen species, resulting in the initiation of oxidative stress and perturbation of retinoic acid signaling and alterations in the expression of growth-regulatory vasculogenic factors and their receptors.

A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke.

Oxidative stress is a major source of injury from cerebral ischemia and reperfusion. We hypothesized that a catalytic antioxidant AEOL 10150 [manganese (III) meso-tetrakis (di-N-ethylimidazole) porphyrin] would attenuate changes in brain gene expression in a mouse model of transient middle cerebral artery occlusion (MCAO). C57BL/6J mice were subjected to either sham surgery or 60 min of right MCAO. AEOL 10150 or phosphate-buffered saline was given intravenously 5 min after onset of reperfusion (n = 6 per group). Six hours later, parenchyma within the MCA distribution was harvested. RNA from the six brains in each group was pooled and mRNA expression determined using an Affymetrix murine MG_U74A v. 2.0 expression microarray. Each experiment was performed three times. The largest changes in expression occurred in stress response and inflammatory genes such as heat shock protein, interleukin-6, and macrophage inflammatory protein-2. Treatment with AEOL 10150 attenuated only the increase in expression of inflammatory genes. This suggests that AEOL 10150 protects brain by attenuating the immune response to ischemia and reperfusion.

The hair growth promoting effect of Sophora flavescens extract and its molecular regulation.

In search of natural extracts for hair growth, we found that the extract of dried root of Sophora flavescens has outstanding hair growth promoting effect. After topical application of Sophora flavescens extract onto the back of C57BL/6 mice, the earlier conversion of telogen-to-anagen was induced. The growth of dermal papilla cells cultured in vitro, however, was not affected by Sophora flavescens extract treatment. RT-PCR analysis showed that Sophora flavescens extract induced mRNA levels of growth factors such as IGF-1 and KGF in dermal papilla cells, suggesting that the effects of Sophora flavescens extract on hair growth may be mediated through the regulation of growth factors in dermal papilla cells. In addition, the Sophora flavescens extract revealed to possess potent inhibitory effect on the type II 5alpha-reductase activity. Taken together, these results suggest that Sophora flavescens extract has hair growth promoting potential and can be used for hair growing products.

Global chimeric exchanges within the intracellular face of the bradykinin B2 receptor with corresponding angiotensin II type Ia receptor regions: generation of fully functional hybrids showing characteristic signaling of the AT1a receptor.

The intracellular (IC) face of the G-protein coupled receptors (GPCR), bradykinin (BK) B2 and angiotensin (AT) 1a, is similar in sequence homology and in size. Both receptors are known to link to Galphai and Galphaq but differ markedly in a number of physiologic actions, particularly with respect to their hemodynamic action. We made single as well as multiple, global replacements within the IC of BKB2R with the corresponding regions of the AT1aR. When stably transfected into Rat-1 cells, these hybrid receptors all bound BK with high affinity. Single replacement of the intracellular loop 2 (IC2) or the distal 34 residues of the C-terminus (dCt) with the corresponding regions of AT1aR resulted in chimera, which turned over phosphotidylinositol (PI) and released arachidonic acid (ARA) as WT BKB2R. In contrast, incorporation of the AT1aR IC3 in a single replacement abolished signal transduction. However, the simultaneous exchange of IC2 and IC3 of BKB2R with AT1aR resulted in a receptor responding to BK with PI turnover and ARA release approximately 4-fold greater than WT BKB2R. Likewise, the simultaneous replacement of IC2 and dCt resulted in a 2.8- and 1.6-fold increase in PI turnover and ARA release, respectively. In contrast, the dual replacement of IC3 and dCt could not overcome the deleterious effects of the IC3 replacement, resulting in very low PI activation and ARA release. Replacement of all three IC domains (IC2, IC3, and dCt) resulted in PI closer to that of AT1aR than BKB2R. The uptake of the receptor chimeras was similar to that of WT BKB2R with the exception of the IC3/dCt dual mutant, which exhibited very poor internalization (18% at 60'). When transfected into Rat-1 cells, the AT1aR markedly increased the expression of connective tissue growth factor (CTGF) mRNA, while BK slightly decreased it. The dual IC2/dCt and triple IC2/IC3/dCt hybrids both upregulated CTGF mRNA in response to BK. These results show that the IC face of the BKB2R can be exchanged with that of AT1aR, producing hybrid receptors, which take on the functional characteristics of AT1aR. The characterization of the chimera with stepwise replacement of the IC domains should allow for assignment of specific roles to the individual loops and C-terminus in the signaling and internalization of the BKB2R and facilitate the generation of a receptor with BKB2R binding and AT1aR function.

Granulin precursor gene: a sex steroid-inducible gene involved in sexual differentiation of the rat brain.

The mechanisms of sexual differentiation of the brain by sex steroids seem to be conserved throughout the mammalian species, although there may be some species differences. In rats, sex-dependent differentiation of the brain occurs in a sex steroid-dependent manner during the perinatal period known as the critical period. Androgen exposure during the perinatal period results in the development of structural and functional sexually dimorphic characteristics in the brain; the absence of testicular androgen leads the central nervous system to develop passively in a primarily female fashion, while the presence of androgen induces the masculinization of the brain. We attempted to characterize sex steroid-inducible genes that are involved in the sexually dimorphic function of the brain. Following the cDNA subtraction between hypothalami of 5-day-old intact and neonatally androgenized female rats, a granulin (grn) precursor gene was identified. The grn gene encodes a 6-kDa polypeptide known as a growth modulating factor of epithelial cells in vitro. Exogenous estrogen, as well as androgen, induced grn gene expression in the neonatal hypothalamus. In the brain of a 5-day-old male rat, grn mRNA was expressed in the ventromedial hypothalamic nucleus and the arcuate nucleus of the hypothalamus. Throughout the critical period for sexual differentiation of the brain, grn gene expression remained high in males, while in females it gradually decreased. Antisense oligodeoxynucleotide (ODN) complementary to grn mRNA was synthesized and infused into the third ventricle of male rats at 2 days of age. Two different control treatments were used; the first consisted of a control sequence ODN that had virtually no homology to known mRNAs, and the second consisted of vehicle alone. After maturation, the subject animals that were treated with antisense ODN of grn displayed significantly lower scores than the control males in various parameters assessing sexual behavior, i.e., mount, intromission, and ejaculation. The present results suggest that the grn gene, the expression of which is induced by sex steroids in the neonatal hypothalamus, plays a crucial role in the functional masculinization of the rat brain.

Critical aspects of tissue-engineered therapy for bone regeneration.

Recent advances in bone tissue engineering are established on the understanding of an engineered scaffold, the molecular milieu within the osteogenic site, and the cell(s) predisposed to an osteogenic lineage. Advances in the incorporation of a generative vehicle into a skeletal defect require temporal and spatial distribution of the scaffold, growth factor, and cell compatible with enhanced bone healing. Monitoring events culminating in osteogenesis has focused on phenotypic and intracellular indicators. Phenotypic and intracellular indicators include the presence of receptors and intracellular signals that enable cell proliferation and differentiation. Progress in the areas of scaffold design, growth factor utilization, bone cell lineage, and intracellular signaling are reviewed.

Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells.

BACKGROUND & AIMS: Epidermal growth factor (EGF)-like growth factors are induced after acute gastric injury and may play an important role in mucosal repair. However, the mechanisms that trigger these growth factors are poorly understood. We determined the role of EGF receptor (EGFR) in stress-induced expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric epithelial cell line (RGM1 cells). METHODS: RGM1 cells were transfected with a plasmid containing complementary DNA encoding a dominant-negative human EGFR (HERCD533). Cells were treated with hydrogen peroxide (0-400 micromol/L) or sorbitol (600 mmol/L). Tyrosine phosphorylation of EGFR was determined by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. HB-EGF messenger RNA and protein were determined with Northern and Western blotting, respectively. Cell growth was evaluated by cell number and [(3)H]thymidine incorporation. RESULTS: Oxidative stress and osmotic stress induced tyrosine phosphorylation of EGFR within 2 minutes, followed by a marked increase in HB-EGF and amphiregulin transcripts in RGM1 cells. Introduction of HERCD533 into the cells inhibited not only tyrosine phosphorylation of EGFR but also growth response to EGF. Furthermore, oxidative stress-induced HB-EGF messenger RNA expression was impaired in HERCD533-expressing cells. CONCLUSIONS: EGFR plays a crucial role in the stress-induced expression of EGF-like growth factors in gastrointestinal epithelial cells.

Molecular cloning and characterization of CHM1L, a novel membrane molecule similar to chondromodulin-I.

Chondromodulin-I (ChM-I) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. In the present study, we identified a novel ChM-I like molecule, designated ChM1L. Cloning of full length cDNAs of human, mouse, and rat ChM1L revealed that ChM1L encodes 317 amino acids novel type II transmembrane protein. ChM1L protein was expressed on the cell surface as N-glycosylated and non-N-glycosylated protein with molecular mass of 45 and 40 kDa, respectively. In adult mouse tissues, ChM1L mRNA was highly expressed in eye, skeletal muscle, and whole rib. The temporal pattern of ChM1L mRNA was examined using whole embryo at day 10 to 19 of gestation. After day 11, ChM1L mRNA was detected and its level was progressively elevated in association with development of mouse embryo. These data suggest that ChM1L is a novel membrane molecule which is similar to ChM-I that plays a regulatory role in eye, skeletal muscle, and development of embryo.

Molecular cloning of delta-4, a new mouse and human Notch ligand.

Complementary DNAs encoding a previously unidentified mouse Notch ligand and its human ortholog were isolated. The new Notch ligand contains a signal sequence, a DSL domain, eight epidermal growth factor-like repeats, a transmembrane domain, and an intracellular region, all of which are characteristics of members of the Delta protein family. The new protein was therefore designated Delta-4. Several previously unidentified sequences in both the extracellular and intracellular regions were shown to be conserved among vertebrate Delta proteins. The tissue distribution of Delta-4 mRNA resembles that previously described for Notch-4 (Int-3) transcripts. However, in situ hybridization with mouse lung revealed that Delta-4 mRNA is abundant in squamous alveolar cells that neighbor endothelial cells; Notch-4 expression is largely restricted to the latter cell type. Soluble forms of the extracellular portion of Delta-4 inhibit the apparent proliferation of human aortic endothelial cells, but not human pulmonary arterial endothelial cells.

Induction of granulin precursor gene expression by estrogen treatment in neonatal rat hypothalamus.

Our previous research has demonstrated that androgen treatment during the perinatal period increases granulin (grn) precursor mRNA levels in the neonatal rat hypothalamus. To elucidate whether exogenous estrogen increases grn mRNA in the neonatal hypothalami, expression of grn gene in the neonatal hypothalamus was studied by the competitive reverse transcription-polymerase chain reaction method. At 6 and 10 days of age, grn gene expression was significantly increased in the hypothalamus of pups whose dam has been dietarily administrated ethinyl estradiol from day 15 of gestation to the day of sampling. The subcutaneous injection of estradiol benzoate to neonatal rats at 2 days of age significantly increased grn gene expression on day 10. It was shown that estrogen, as well as androgen, was able to induce grn gene expression in the neonatal hypothalamus.

Genetic basis of susceptibility to environmentally induced neural tube defects.

Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain-dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate-regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.

Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues.

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

Identification of a common receptor for three types of rat cytokine-induced neutrophil chemoattractants (CINCs).

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/macrophage inflammatory protein-2 (MIP-2), members of the CXC chemokine family, are potent chemotactic factors for neutrophils. In order to identify the receptor for CINCs, rat CXC chemokine receptor 2 (CXCR2) was cloned and expressed in HEK293 cells. CINC-1, CINC-2 and CINC-3 induced calcium mobilizations dose-dependently in CXCR2-transfected cells, whereas formyl-methionyl-leucyl-phenylalanine (FMLP) did not. CINC-3 induced enhancement of cytoplasmic calcium concentration more potently than CINC-1 and CINC-2, and desensitized calcium transients induced by CINC-1 and CINC-2, which were essentially identical to those observed in rat neutrophils. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of three types of CINCs almost completely. The mutant CINC-3, whose amino-terminal amino acid sequence (SELR) was replaced to AAR, lost chemotactic activity of its own but inhibited that of CINC-1 and CINC-2 potently, and that of CINC-3 weakly. The results indicate that rat CXCR2 on neutrophils is the unique receptor for all three types of CINCs, and CINC-1/-2 and CINC-3 exert different biological activities through the common receptor.