The objective assessment of the effects on cognition functioning among military personnel exposed to hypobaric-hypoxia: A pilot fMRI study.

OBJECTIVE: To identify regions of the brain affected during cognitive working memory during tasks to assess attention, planning and decision making among military aviation personnel who have chronic intermittent exposure to high altitude environment. METHOD: A case-control study was conducted in the Universiti Putra Malaysia among eight military personnel, four of whom had chronic intermittent exposure to high altitude training. They were divided into two groups, chronic intermittent exposure group (CE) (n=4) and a control group (n=4). They underwent a task-based functional magnetic resonance imaging (fMRI) that utilised spatial working memory task to objectively evaluate the neural activation in response to the Tower of London paradigm. Each correct answer was given a score of one and the maximum achievable score was 100%. RESULTS: A consecutive dichotomised group of CE (4/8) and control (4/8) of age-matched military aviation personnel with a mean age of 37.23±5.52 years; showed significant activation in the right middle frontal gyrus (MFG). This in turn was positively correlated with response accuracy. A significant difference in the response accuracy was noted among both the groups at p<0.05. CONCLUSION: At the minimum results of power analysis of this preliminary fMRI study, our group of aviation personnel who had chronic intermittent exposure to hypobaric hypoxic environment, did not have any significant decrease in cognitive function namely attention, decision-making and problem solving compared to controls during a working memory task.

Advanced Merkel cell carcinoma of the lower extremity treated with surgery and isolated pelvic and limb perfusion using Melphalan: A case of unexpected long-term survival.

INTRODUCTION: Merkel cell carcinoma (MCC) is a rare, neuroendocrine skin tumor, with high frequency of locoregional recurrence, metastases, and poor prognosis. Locoregional MCC recurrence in the extremities can pose considerable treatment challenges. We report a case of long-term survival in a female patient with recurrent MCC of the leg, treated with surgery and locoregional chemotherapy. PRESENTATION OF CASE: A 73-year-old female with cirrhosis and hepatitis C, developed cutaneous MCC in the left inferior limb. This patient initially received surgical treatment, with subsequent negative sentinel lymph-node biopsy in another center, one-month prior recovery in our department, and arrived with 4 new limb nodules, cranially to the previously treated area, without distant metastases or inguinal lymph node recurrence. This patient was not eligible for immunotherapy due to active hepatitis upon treatment with NS5B inhibitors, or eligible for systemic chemotherapy or radiotherapy due to severe neutropenia and was, therefore, subjected to surgical resection combined with Isolated Pelvic and Limb Perfusion (IPLP) with Melphalan. Histological evaluation confirmed MCC diagnosis and during the following 4 months, she developed further locoregional recurrences with homolateral inguinal lymph node involvement and was subjected to two additional rounds of surgery plus IPLP. DISCUSSION: All procedures were tolerated, systemic toxicities were temporary and subsequent clinical and radiological follow-up, following the last combined treatment, indicated that this patient was still alive and disease-free, at 56 months. CONCLUSION: In this case, surgery combined with locoregional Melphalan chemotherapy was an effective and repeatable treatment for recurrent MMC and resulted in unexpected long-term survival.

NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis.

We report a novel pro-apoptotic function for nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) receptor in sensitizing TRAIL (TNF-related apoptotis-inducing ligand)-resistant SH-SY5Y neuroblastoma (NB) cells to TRAIL-induced apoptosis, resulting in the abrogation of anchorage-independent tumourigenic growth in vitro. We show that the TRAIL-resistant SH-SY5Y phenotype is cFLIP (cellular FLICE-like inhibitory protein) dependent and not due to low-level functional TRAIL receptor or caspase expression or an inhibitory equilibrium between functional and decoy TRAIL receptors or B-cell lymphoma 2 (Bcl-2) and BH3-only (Bcl-2 homology domain 3-only) family proteins. NGF sensitization of SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA expression, activation and subsequent sequestration of cFLIP. This reduces cFLIP recruitment to TRAIL-activated death receptors and increases the recruitment of caspase-8, leading to TRAIL-induced, caspase-dependent, type II apoptosis via the intrinsic mitochondrial pathway. This effect was temporary, inhibited within 6 h by nuclear factor-κ binding (NF-κB)-mediated increase in myeloid cell leukaemia-1 (Mcl-1) expression, abrogated by transient cFLIP or B-cell lymphoma-extra large (Bcl-xL) overexpression and optimized by NF-κB and Mcl-1 inhibitors. This novel mechanism adds an important pro-apoptotic immunological dimension to NGF/TrkA interaction that may not only help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB, but also provides a novel potential pro-apoptotic therapeutic use for NGF, TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance.

The neuroblastoma tumour-suppressor TrkAI and its oncogenic alternative TrkAIII splice variant exhibit geldanamycin-sensitive interactions with Hsp90 in human neuroblastoma cells.

Hsp90 chaperones stabilize many tyrosine kinases including several oncogenes, which are inhibited or induced to degrade by the Hsp90 inhibitor geldanamycin (GA). As a consequence, GA has been developed for future chemotherapeutic use in several tumour types including neuroblastoma (NB). Alternative splicing of the neurotrophin receptor tyrosine kinase TrkA may have a pivotal function in regulating NB behaviour, with reports suggesting that tumour-suppressing signals from TrkA may be converted to oncogenic signals by stress-regulated alternative TrkAIII splicing. Within this context, it is important to know whether Hsp90 interacts with TrkA variants in NB cells and how GA influences this. Here, we report that both TrkAI and TrkAIII are Hsp90 clients in human NB cells. TrkAI exhibits GA-sensitive interaction with Hsp90 required for receptor endoplasmic reticulum export, maturation, cell surface stabilization and ligand-mediated activation, whereas TrkAIII exhibits GA-sensitive interactions with Hsp90 required for spontaneous activity and to a lesser extent stability. We show that GA inhibits proliferation and induces apoptosis of TrkAI expressing NB cells, whereas TrkAIII reduces the sensitivity of NB cells to GA-induced elimination. Our data suggest that GA-sensitive interactions with Hsp90 are critical for both TrkAI tumour suppressor and TrkAIII oncogenic function in NB and that TrkAIII expression exerts a negative impact on GA-induced NB cell eradication, which can be counteracted by a novel TrkAIII-specific peptide nucleic acid inhibitor.

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.

Transcriptional up-regulation of matrix metalloproteinase-9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor kappaB elements.

Spontaneous epithelial (S) to neuroblast (N) conversion enhanced the capacity of SK-N-SH neuroblastoma (NB) cells to invade reconstituted basement membrane in vitro. This involved a switch to matrix metalloproteinase (MMP) activity, in particular MMP-9, and was associated with the induction of MMP-9 expression. N-type-specific MMP-9 expression was herbimycin A inhibitable tyrosine kinase (possibly c-src) dependent and was regulated transcriptionally through GT-box (-52), and nuclear factor kappaB (NFkappaB; -600) elements within the MMP-9 gene. GT-box function was associated with elevated levels of specific nuclear GT-box binding complexes in N-type cells. NFkappaB function was associated with specific p50- and p65-containing nuclear NFkappaB binding complex(es). No function could be attributed to the proximal AP-1 (-79) element, and minimal function was attributed to the SP-1 (-560), ets (-540), or distal AP-1 (-533) elements. This was despite elevated levels of specific junD/fra-1 containing proximal AP-1 element binding complex(es) in N-type cells. Our data highlight a pivotal role for the GT-box, in concert with the NFkappaB element, in the transcriptional up-regulation of MMP-9 expression during spontaneous S to N phenotype conversion by SK-N-SH cells involved in enhanced basement membrane invasivity.

Tissue inhibitor of metalloproteinase-2 protection of matrix metalloproteinase-2 from degradation by plasmin is reversed by divalent cation chelator EDTA and the bisphosphonate alendronate.

The degradation of tissue inhibitor of metalloproteinase (TIMP)-free matrix metalloproteinase (MMP)-2 to proteolytically inactive fragments by plasmin was inhibited in equimolar mixtures of purified TIMP-2 and TIMP-free MMP-2 and was not observed in purified MMP-2-TIMP-2 complexes. Divalent cation chelators EDTA and sodium Alendronate did not inhibit plasmin degradation of TIMP-free MMP-2 but reversed the ability of TIMP-2 to protect MMP-2 from degradation by plasmin. Our data confirm a role for plasmin in the clearance of TIMP-free MMP-2, identify a pivotal role for TIMP-2 in regulating MMP-2 longevity in plasmin-containing environments, and highlight a novel therapeutic use for chelators of divalent cations, including the bisphosphonate Alendronate, in the reversal of TIMP-2 protection of MMP-2 from degradation by plasmin. We propose that these observations are relevant to pathologies that are dependent upon plasmin and MMP-2 activity (e.g., tumor invasion and metastasis).

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent.

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-alphaVbeta3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology.

Transforming growth factor-beta1 enhances the invasiveness of human MDA-MB-231 breast cancer cells by up-regulating urokinase activity.

Transforming growth factor-beta (TGFbeta1) enhances human MDA-MB-231 breast tumour cell invasion of reconstituted basement membrane in vitro but does not inhibit proliferation of this cell line. In contrast to basal invasion, which is plasmin-, urokinase (uPA)-, tissue-type plasminogen activator (t-PA)-, matrix metalloproteinase (MMP)-9- and TIMP-1-inhibitable MMP-dependent, TGFbeta1 enhanced-invasion is dependent upon plasmin and uPA activity but does not appear to involve t-PA-, MMP9- or TIMP-1-inhibitable MMPs, as judged by inhibitor studies. Enhanced invasion is associated with increased u-PA, UPAR, PAI-1, MT-MMP-1, MMP-9 and TIMP-1 expression; with reduced t-PA, MMP-1 and MMP-3 expression; and with the induction of membrane MMP-9 association. The net result of these changes includes increased secreted, but not membrane-associated, uPA levels and activity and reduced secreted levels of plasmin and APMA-activatable gelatinolytic, collagenolytic and caseinolytic MMP activity but no change in membrane-associated gelatinolytic activity, despite increased MT-MMP-1 expression and MMP-9 membrane association. TGFbeta1 does not induce MMP-2 expression. Our data indicate that TGFbeta1 can promote the malignant behaviour of MDA-MB-231 cells refractory to TGFbeta1-mediated proliferation control by enhancing their invasive capacity. We suggest that this results from the action of a uPA/plasmin-dependent mechanism resulting from stimulation of uPA expression, secretion and subsequent activity, despite elevated PAI-1 inhibitor levels.

Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induction of transcription by IFN-gamma.

We describe the molecular features of the interferon (IFN)-gamma-mediated transcription of the human intercellular adhesion molecule (ICAM-1) gene. We identified putative IFN-gamma-activated sites (GAS) distributed throughout a large segment of the ICAM-1 promoter (4.0 kb region). Using computer-assisted search, these sequences were similar to potential IFN-gamma responsive elements that have a core sequence 5'-TTNCNNNAA-3'. In this report we show that in the ICAM-1 promoter a GAS site is located at -115 from the translation initiation site, and binds with strong affinity to IFN-gamma-activated Signal Transducers and Activators of Transcription (STAT1) homodimers. The same sequence is responsible for the IFN-gamma-mediated transcription of the ICAM-1 gene. Moreover, we present evidence that a more distal GAS element that maps at -2787 from the translation initiation site, binds IFN-gamma-activated STAT1 dimers with lower affinity. Multimeric copies of such GAS sequence inserted into a tkCAT minimal promoter can drive transcription, demonstrating that the -2787 bp GAS element has an independent functional activity upon binding of IFN-gamma-activated STAT1 proteins as documented by in vitro binding assays. Furthermore, using recombinant ICAM-CAT mutants, we show that, in vivo, the -2787 GAS, but not a mutagenized -2787 GAS site, when coupled to the more proximal -115 GAS element, has an additive effect in enhancing the IFN-gamma-mediated transcription of ICAM-1 promoter. Nevertheless, using a recombinant construct bearing the wild type -2787 GAS element and a mutagenized -115 GAS element, we could not detect any transcription after transfection of U937 recipient cells, suggesting that the -2787 bp GAS element is not sufficient as such for gene activation, but can cooperate with its cognate proximal sequence to give full function to the ICAM-1 promoter during the IFN-gamma response. Taken together these data provide evidence that two GAS sites are required for the full potential activity in the mechanism of ICAM-1 gene activation by IFN-gamma.

Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator.

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation.

Transcriptional regulation of intercellular adhesion molecule 1 by phorbol ester in human neuroblastoma cell line SK-N-SH involves jun- and fos-containing activator protein 1 site binding complex(es).

In this study, the regulatory elements involved in ICAM-1 transcriptional response to phorbol ester (12-0-tetradecanoylphorbol-13-acetate; TPA) have been investigated in the human neuroblastoma cell line, SK-N-SH. TPA induced intercellular adhesion molecule 1 (ICAM-1) protein expression in SK-N-SH cells within 24 h of treatment as judged by indirect immunofluorescence. Basal ICAM-1 mRNA levels were barely detectable in untreated SK-N-SH cells but were induced by TPA to a maximal level with 4 h and were reduced thereafter. Analysis of the 5' promoter sequence of ICAM-1 revealed two regions that functioned equally in the TPA induction of ICAM-1 transcription. The first region (-145 to -227) contained a nuclear factor-kappa B (NF kappa B) element. The second region (-316 to -390) contained a putative TPA-responsive element (TRE; TGATTCA) and a TATA box. Deletion and point mutation of the latter region indicated that the TRE was indeed the functional element within this region and acted fully and independently of all other elements including the TATA box at position -352. This TRE bound TPA induced specific nuclear complexes in vitro containing junD, c-jun, c-fos, and fra2 but not cAMP-responsive element binding/activating transcription factor family proteins. ICAM-TRE binding activity was induced within 30 min following TPA treatment. This preceded the appearance of ICAM-NF kappa B site binding activity. Cotransfection of c-jun and c-fos expression vectors into SK-N-SH cells induced transactivation from ICAM-1 promoter constructs containing the intact but not mutated TRE site. Primer extension analyses revealed that TPA had induced transcription exclusively at two sites -40 and -41 bp upstream of the translation start site. These data show that the ICAM-TRE and its cognate jun- and fos-containing transcription factors play a predominant role in the transcriptional response of ICAM-1 to the protein kinase C activator TPA in SK-N-SH cells.

Identification of plasminogen in Matrigel and its activation by reconstitution of this basement membrane extract.

Matrigel, a basement membrane (BM) extract of the Engelbreth-Holm-Swarm (EHS) sarcoma, used in tumor invasion assays, was found to contain plasminogen. Plasminogen was identified, using Western blot analysis and casein zymograms, by comparison with human plasminogen. matrigel contained approximately 20-100 ng of plasminogen per 100 micrograms of protein as determined by these assays. Matrigel reconstitution and incubation at 37 degrees C caused activation of plasminogen, which was serine protease dependent and involved tissue plasminogen activator (tPA) as an anti-tPA antibody which inhibited activation. This reconstitution and incubation also caused leupeptin-inhibitable degradation of Matrigel components as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Degradation of the BM extract copolymerized in zymograms was caused by human plasminogen and plasminogen in the Matrigel. Maximal plasmin activity, following incubation of Matrigel at 37 degrees C for 16 h, was equivalent to approximately 10 ng of purified plasmin using the plasmin substrate D-Val-Leu-Lys p-nitroanilide. matrigel, therefore, contained all the components of the plasmin-generating system, including plasminogen. The plasmin generated degraded Matrigel components and exogenous substrates. Our data suggest that, since this tumor BM acts as a reservoir for enzymes of the plasmin-generating system, caution should be taken by investigators interpreting data concerning the effects of Matrigel on cell behavior and, in particular, cellular invasion.

Prostaglandin E2 inhibits the interleukin-2 promoter activity through down-regulation of the Oct-dependent transcription of the octamer motif.

Prostaglandins, mainly those of the E series (PGE), are modulators of immune responses. Indeed PGE2 inhibits T cell activation and the transcription of the interleukin-2 (IL-2) gene, the major T cell growth factor. We observed that PGE2 inhibits IL-2 promoter transcription activity by interfering with signals activating the (-96 to -66 bp) octamer motif. This motif binds Oct-1 and Oct-2 as well as the phorbol ester and calcium ionophore-inducible jun and fos AP-1 factors. The PGE2-dependent down-modulation is observed in the presence of either the endogenous transacting factor Oct-1 or the exogenously expressed Oct-2. PGE2 does not regulate octamer function by influencing the jun and fos mRNA or Oct-1 protein levels or their DNA-binding abilities. Functional dissection of the octamer motif, through mutations of either the AP-1 or the octamer sites, revealed that the AP-1 site is dispensable for PGE2-dependent inhibition which instead may occur through the interference with the Oct-mediated transactivation of the octamer element. Our data suggest that the Oct-octamer interaction is a novel target of the PGE2-induced down-regulation of the IL-2 promoter.

Transcriptional modulation of the human intercellular adhesion molecule gene I (ICAM-1) by retinoic acid in melanoma cells.

Retinoids play an important role as differentiating agents in a variety of normal and neoplastic cells and have been reported to induce ICAM-1 levels in melanomas, a phenomenon that we confirm in this paper. The effects of retinoids on gene expression usually involve the binding of specific retinoic acid receptor trans-acting factors (RARs) with their ligands, which then interact with specific target sites, the retinoic acid responsive elements (RAREs) present in the promoters of responsive genes. In the case of ICAM-1, we have cloned and analyzed the proximal regulatory region of the human gene. We show that the ICAM-1 promoter is RA-inducible, that it contains a putative consensus RARE (GGGTCATCGCCCTGCC), which binds in vitro RAR alpha complemented with RXRs, and that mutation of the RARE abrogates promoter responsiveness to RA. These studies allow ICAM-1 to be added to the list of genes transcriptionally activated by RA acting through an RARE element.

Follicle-stimulating hormone increases the expression of tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 and induces TIMP-1 AP-1 site binding complex(es) in prepubertal rat Sertoli cells.

Primary cultures of prepubertal rat Sertoli cells secrete two major tissue inhibitors of metalloproteinases: TIMP-1 (M(r) 28K) and TIMP-2 (M(r) 21 K). FSH stimulated Sertoli cell TIMP-1 and TIMP-2 activity in a time- and dose-dependent manner and also stimulated TIMP-1 and TIMP-2 protein and messenger RNA levels. These effects were mimicked by the cAMP analog, 8-bromo-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The protein kinase C activating phorbol ester phorbol myristate acetate (TPA) stimulated TIMP-1 but not TIMP-2 activity and messenger RNA levels. Cycloheximide and actinomycin-D inhibited basal TIMP-1 and TIMP-2 activity and inhibited the ability of FSH, 8-bromo-cAMP, and TPA to stimulate TIMP activity. The protein kinase A (PKA) inhibitor AMP Rp isomer did not affect basal TIMP-1 and TIMP-2 activity or TPA-stimulated TIMP-1 activity. However, the PKA inhibitor markedly reduced FSH and 3-isobutyl-1-methylxanthine stimulation of TIMP-1 and TIMP-2 activity. FSH, 8-bromo-cAMP, and TPA stimuli induced DNA binding complexes capable of binding to a TIMP-1 AP-1 site consensus sequence oligonucleotide. The AP-1 site binding complex(es) induced by all three treatments reacted with antibodies directed broadly against fos and jun protooncogene families and against the specific family members c-fos, junB, and junD but not c-jun proteins. Constitutive cAMP response element binding activity capable of binding an artificial cAMP response element binding site oligonucleotide was demonstrated in Sertoli cell nuclear extracts. This activity was minimally modulated by FSH, 8-bromo-cAMP, or TPA treatment. In summary, Sertoli cells secrete TIMP-1 and TIMP-2 that can be coordinately up-regulated by FSH through a cAMP, PKA-dependent pathway. a convergence of TPA, FSH, and cAMP mediated signals in prepubertal Sertoli cells may occur with the induction of specific AP-1 site binding complex(es) containing jun and fos proteins. Our data suggest that FSH stimulation of TIMP-2 expression may be regulated independently to that of TIMP-1. We propose that the ability of FSH to stimulate Sertoli cell TIMP activity suggests a central role for this hormone in the control of extracellular matrix turnover during testicular development at the level of metalloproteinase inhibition.

Positive and negative regulation of the composite octamer motif of the interleukin 2 enhancer by AP-1, Oct-2, and retinoic acid receptor.

The differentiating agent retinoic acid (RA) has been previously reported to interfere with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)/Ca2+-induced signals for the regulation of the -96 to -66-bp octamer motif found in the enhancer for the interleukin (IL)-2 gene, which encodes a major T lymphocyte growth factor. The IL-2 octamer motif is a composite cis-element which binds Oct-1 and Oct-2 as well as a TPA/Ca2+-inducible nuclear factor, previously termed octamer-associated protein (OAP40). We show here that Oct-2, despite the presence of an active transcriptional activation domain, requires TPA/Ca2+-induced signals to strongly transactivate the IL-2 octamer motif in Jurkat T cells. This Oct-2-dependent transactivation is inhibited by RA. The presence of an intact COOH-terminal domain of Oct-2 contributes to both TPA/Ca2+-induced transactivation and the RA-mediated repression. We also show that both Fos and Jun components of the AP-1 factors participate in the OAP40 complex. Furthermore, transfected c-jun, jun-B, jun-D, c-fos, or Fos-B expression vectors partially substitute for TPA and Ca2+ and cooperate with Oct-2 for the transactivation of the combined OAP/octamer cis-element. Mutations of the genuine octamer-binding site abrogate both the binding of Oct-1 and Oct-2 and the TPA/Ca2+-induced transactivation of the OAP/octamer motif. OAP confers to Oct-2 responsivity to both TPA/Ca2+ and RA, since specific mutations of the AP-1/OAP-binding site significantly reduce the transactivation by Oct-2 in response to TPA and Ca2+ and abolish the inhibition by RA. Furthermore, retinoic acid receptor (RAR) alpha is able to inhibit in vitro the formation of the complex between the nuclear AP-1/OAP and its specific binding site, resulting in the interference with Oct-2-dependent cis-regulatory function of this AP-1 element. Therefore, we propose that the TPA/calcium-activated AP-1/OAP element is the main target of positive or negative regulatory signals influencing the IL-2 octamer motif, through synergism with Oct-2 and antagonism by RAR.

An early response of an AP1-junD complex during T-cell activation.

Activated T-cell extracts contain an activity (T-AP1) composed of at least two dissociable protein components which bind to the AP1 consensus sequence in the enhancer of the gibbon ape leukemia virus (GALV)-LTR (GALV-TRE). This activity is inducible by 12-O-tetradecanoyl-phorbol-14-acetate (TPA) even in the presence of protein synthesis inhibitors. Although one component of this complex (CORE) is related immunologically and biochemically to junD, it nevertheless displays significant biochemical properties which distinguish CORE from recombinant junD. The second component of the complex, flowthrough, interacts more efficiently with CORE than with recombinant junD. GALV-TRE enhancer activity is increased within 2 h in vivo with T cells treated with TPA in the presence of protein synthesis inhibitors; this increase in enhancer activity is paralleled by the increased GALV-TRE-mediated transcriptional activity present in extracts of these cells. Purified T-cell junD activates GALV-TRE-driven RNA synthesis in vitro. The rapidity and the protein synthesis-independent nature of TPA-induced T-AP1 activation suggests that this complex is involved in the earliest stages of T-cell activation.

Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription.

Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential.