Visualization of the earliest steps of gammadelta T cell development in the adult thymus.

The checkpoint in cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls cell development during the penultimate CD4-CD8- stage. Passage through this checkpoint required the assembly of TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.

Differential Modulation of Transcriptional Activity of Estrogen Receptors by Direct Protein-Protein Interactions with the T Cell Factor Family of Transcription Factors

Two major signaling pathways, those triggered by estrogen (E(2)) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ER(alpha) and ER(beta) are activated by binding E(2) and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E(2). For mutants of the former promoter, the stimulatory effects of ER(alpha)/E(2) can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E(2). Direct interaction between ERs and Tcfs either at the Tcf/ER(alpha)-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivo in this tissue.

Osteoclastogenesis induced by sRANKL and M-CSF from the non-adherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF

During bone development and repair, angiogenesis, osteogenesis and bone remodelling are closely associated processes that share some common mediators. In the present study non-adherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, 300 ng/ml) and rhVEGF (0, 25 ng/ml) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/ml showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/ml) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/ml rhBMP-2. However, rhVEGF induced resorptive function was inhibited by 30 ng/ml and 300 ng/ml rhBMP-2 at a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis, exact mechanisms are yet to be elucidated.

Calcium signaling in ocular arterioles

Local control of blood flow to the photoreceptors and associated neurons in the retina is largely achieved through changes in tone within the choroidal and retinal arterioles. This is primarily achieved through changes in [Ca2+] within the smooth muscle of these vessels, which regulates cell contraction and vascular constriction. Here we review some aspects of the cell physiology involved in these Ca2+-signaling processes, with particular emphasis on the molecular mechanisms involved. Ca2+-influx across the plasma membrane can occur via a variety of Ca2+-channels, including voltage-operated, store-operated, and receptor-operated channels. Ca2+ may also be released from intracellular stores via RyR-, or IP3R-gated channels in the SR membrane. Using high-speed confocal Ca2+-imaging, we have also demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca2+-sparks and more global Ca2+-waves and oscillations. These signals may be specifically and differentially targeted, for example, to Ca2+-sensitive ion channels (stimulus-excitation coupling), or pathways regulating contraction (stimulus-contraction coupling). Exploring the role of changes in such targeting in disease states will provide exciting opportunities for future research.

Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells.

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of approximately 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 +/- 0.02-fold (P <0.005) relative to control with approximately 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.

HIF-1 and NF-kappaB-mediated upregulation of CXCR1 and CXCR2 expression promotes cell survival in hypoxic prostate cancer cells

Hypoxic cancer cells are resistant to treatment, leading to the selection of cells with a more malignant phenotype. The expression of interleukin-8 (IL-8) plays an important role in the tumorigenesis and metastasis of solid tumors including prostate cancer. Recently, we detected elevated expression of IL-8 and IL-8 receptors in human prostate cancer tissue. The objective of the current study was to determine whether hypoxia increases IL-8 and IL-8 receptor expression in prostate cancer cells and whether this contributes to a survival advantage in hypoxic cells. IL-8, CXCR1 and CXCR2 messenger RNA (mRNA) expression in PC3 cells was upregulated in response to hypoxia in a time-dependent manner. Elevated IL-8 secretion following hypoxia was detected by enzyme-linked immunosorbent assay, while immunoblotting confirmed elevated receptor expression. Attenuation of hypoxia-inducible factor (HIF-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity using small interfering RNA (siRNA), a HIF-1 dominant-negative and pharmacological inhibitors, abrogated hypoxia-induced transcription of CXCR1 and CXCR2 in PC3 cells. Furthermore, chromatin-IP analysis demonstrated binding of HIF-1 and NF-kappaB to CXCR1. Finally, inhibition of IL-8 signaling potentiated etoposide-induced cell death in hypoxic PC3 cells. These results suggest that IL-8 signaling confers a survival advantage to hypoxic prostate cancer cells, and therefore, strategies to inhibit IL-8 signaling may sensitize hypoxic tumor cells to conventional treatments.

Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by inhibiting epidermal growth factor receptor pathway.

Our previous studies have shown that overexpression of beta1,4-galactosyltransferase1 (beta1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of beta1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface beta1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface beta1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of beta1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface beta1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.

Cholinergic-induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder.

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca(2+) indicator fluo 4AM. ICC fired Ca(2+) transients in response to stimulation by carbachol (1/10 microM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 microM), an M(3) receptor antagonist, but not by the M(2) receptor antagonist methoctramine (1 microM). The source of Ca(2+) underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 microM) or Ni(2+) (30-100 microM) to block Ca(2+) channels or the removal of external Ca(2+) reduced the amplitude of the carbachol transients. Application of ryanodine (30 microM) or tetracaine (100 microM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 microM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 microM) caused a significant reduction and Xestospongin C (1 microM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca(2+) indicator showed distinctively different patterns of spontaneous Ca(2+) events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca(2+) transients. PMID: 18171995 [PubMed - indexed for MEDLINE]

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells

Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c+FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear factor-kappa B-mediated transcription attenuated IL-8-induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy.

Chemotherapy-Induced CXC-Chemokine/CXC-Chemokine Receptor Signaling in Metastatic Prostate Cancer Cells Confers Resistance to Oxaliplatin through Potentiation of Nuclear Factor-κB Transcription and Evasion of Apoptosis

Constitutive activation of nuclear factor (NF)-kappa B is linked with the intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-kappa B whose expression is elevated in AIPC. This study sought to determine the significance of CXCL8 signaling in regulating the response of AIPC cells to oxaliplatin, a drug whose activity is reportedly sensitive to NF-kappa B activity. Administration of oxaliplatin to PC3 and DU145 cells increased NF-kappa B activity, promoting antiapoptotic gene transcription. In addition, oxaliplatin increased the transcription and secretion of CXCL8 and the related CXC-chemokine CXCL1 and increased the transcription and expression of CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells with CXCL8 potentiated NF-kappa B activation in AIPC cells, increasing the transcription and expression of NF-kappa B-regulated antiapoptotic genes of the Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist, AZ10397767 (Bioorg Med Chem Lett 18:798-803, 2008), attenuated oxaliplatin-induced NF-kappa B activation, increased oxaliplatin cytotoxicity, and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological inhibition of NF-kappa B or RNA interference-mediated suppression of Bcl-2 and survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results further support NF-kappa B activity as an important determinant of cancer cell sensitivity to oxaliplatin and identify the induction of autocrine CXCR2 signaling as a novel mode of resistance to this drug.

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor a (ERa) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERa phosphorylation on Ser118 in response to 17ß-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

The Yin and Yang of vitamin D receptor (VDR) signaling in neoplastic progression: Operational networks and tissue-specific growth control

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1a,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1a,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1a,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1a,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.

Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.