Pinhead signaling regulates mesoderm heterogeneity via the FGF receptor-dependent pathway.

Among the three embryonic germ layers, the mesoderm plays a central role in the establishment of the vertebrate body plan. The mesoderm is specified by secreted signaling proteins from the FGF, Nodal, BMP and Wnt families. No new classes of extracellular mesoderm-inducing factors have been identified in more than two decades. Here, we show that the pinhead (pnhd) gene encodes a secreted protein that is essential for the activation of a subset of mesodermal markers in the Xenopus embryo. RNA sequencing revealed that many transcriptional targets of Pnhd are shared with those of the FGF pathway. Pnhd activity was accompanied by Erk phosphorylation and required FGF and Nodal but not Wnt signaling. We propose that during gastrulation Pnhd acts in the marginal zone to contribute to mesoderm heterogeneity via an FGF receptor-dependent positive feedback mechanism.

Proteomics Analysis of Lymphoblastoid Cell Lines from Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of the progressive degeneration of motor neurons, caused by poorly understood mechanisms for which there is no cure. Some of the cellular perturbations associated with ALS can be detected in peripheral cells, including lymphocytes from blood. A related cell system that is very suitable for research consists of human lymphoblastoid cell lines (LCLs), which are immortalized lymphocytes. LCLs that can be easily expanded in culture and can be maintained for long periods as stable cultures. We investigated, on a small set of LCLs, if a proteomics analysis using liquid chromatography followed by tandem mass spectrometry reveals proteins that are differentially present in ALS versus healthy controls. We found that individual proteins, the cellular and molecular pathways in which these proteins participate, are detected as differentially present in the ALS samples. Some of these proteins and pathways are already known to be perturbed in ALS, while others are new and present interest for further investigations. These observations suggest that a more detailed proteomics analysis of LCLs, using a larger number of samples, represents a promising approach for investigating ALS mechanisms and to search for therapeutic agents. Proteomics data are available via ProteomeXchange with identifier PXD040240.

Vasa nervorum angiogenesis in prostate cancer with perineural invasion.

BACKGROUND: Perineural invasion (PNI) is generally accepted as a major route of cancer dissemination in malignancies associated with highly enervated organs. However, the effect of cancer cells on vasa nervorum remains unknown. We studied this effect in locally advanced prostate cancer, a high-risk feature associated with approximately 20% of prostate cancer specific mortality. METHODS: We used immunohistochemistry for CD34, fibroblast growth factor-2 (FGF-2), FSHR, podoplanin, vascular endothelial growth factor (VEGF), and VEGFR-2 as well as histochemical methods to examine the vasa nervorum of nerves invaded by cancer cells in tissue samples from 85 patients. RESULTS: The percentage of the nerve area occupied by CD34-positive vasa nervorum endothelial cells in nerves with PNI was much higher than in nerves without PNI (7.3 ± 1.2 vs 1.9 ± 0.4; P < 0.001 and 5.8 ± 0.6 vs 1.23 ± 0.8; P < 0.001 in pT3a and pT3b prostate cancer specimens, respectively). In 19/85 of the patients the CD34-positive vasa nervorum microvessels have a thick basement membrane, similar to the vessels in diabetic microangiopathy. This subendothelial layer contains collagen fibers. Vasa nervorum endothelia and Schwann cells express FGF-2 (nuclear localization) and FSHR (plasma membrane and cytoplasmic staining). Prostate cancer cells invading nerves express VEGF, a critical cytokine in tumor angiogenesis. The vasa nervorum of prostatic nerves with PNI did not express detectable levels of VEGFR-2. No podoplanin-positive lymphatic vessels were seen in nerves. CONCLUSION: In locally advanced prostate cancer, PNI of cancer cells is associated with formation of new endoneurial capillaries and changes of vasa nervorum morphology.

Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis.

Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.

Expression of follicle-stimulating hormone receptor in tumor blood vessels.

BACKGROUND: In adult humans, the follicle-stimulating hormone (FSH) receptor is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels. METHODS: We used immunohistochemical and immunoblotting techniques involving four separate FSH-receptor-specific monoclonal antibodies that recognize different FSH receptor epitopes and in situ hybridization to detect FSH receptor in tissue samples from patients with a wide range of tumors. Immunoelectron microscopy was used to detect FSH receptor in mouse tumors. RESULTS: In all 1336 patients examined, FSH receptor was expressed by endothelial cells in tumors of all grades, including early T1 tumors. The tumors were located in the prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary. In specimens obtained during surgery performed to remove tumors, the FSH receptor was not expressed in the normal tissues located more than 10 mm from the tumors. The tumor lymphatic vessels did not express FSH receptor. The endothelial cells that expressed FSH receptor were located at the periphery of the tumors in a layer that was approximately 10 mm thick; this layer extended both into and outside of the tumor. Immunoelectron microscopy in mice with xenograft tumors, after perfusion with anti­FSH-receptor antibodies coupled to colloidal gold, showed that the FSH receptor is exposed on the luminal endothelial surface and can bind and internalize circulating ligands. CONCLUSIONS: FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).

Follicle-stimulating hormone receptor expression in soft tissue sarcomas.

AIMS: In adult humans, the follicle-stimulating hormone receptor (FSHR) is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. Recently, it has been shown that FSHR is expressed selectively on the surface of blood vessels in a wide range of tumours. So far, the expression of FSHR in mesenchymal tumours has not been studied. METHODS AND RESULTS: We performed a semiquantitative evaluation of FSHR protein expression in a large cohort of soft tissue sarcomas (STS; n = 335), including 11 subtypes. FSHR-positive vessels were detected in all sarcoma subtypes analysed. Among liposarcomas, significantly more cases of dedifferentiated liposarcomas (28 of 44) showed FSHR expression compared to well-differentiated liposarcomas (WDLS; four of 21; P < 0.001). Vessels in lipomas (n = 9) and non-neoplastic fat were FSHR-negative. FSHR expression was also detected in tumour cells of all sarcoma subtypes examined, with the lowest incidence in WDLS (three of 21; 14.3%) and the highest frequency in undifferentiated high-grade pleomorphic sarcomas (41 of 60; 68.3%). CONCLUSIONS: These data supplement the previously reported results of FSHR expression in endothelial cells of various cancer types and form a solid basis for further studies of FSHR in mesenchymal neoplasms.

Expression of follicle-stimulating hormone receptor by the vascular endothelium in tumor metastases.

BACKGROUND: The Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human tumors. It was not determined however if FSHR is present in metastases which are responsible for the terminal illness. METHODS: We used immunohistochemistry based on a highly FSHR-specific monoclonal antibody to detect FSHR in cancer metastases from 6 major tumor types (lung, breast, prostate, colon, kidney, and leiomyosarcoma ) to 6 frequent locations (bone, liver, lymph node, brain, lung, and pleura) of 209 patients. RESULTS: In 166 patients examined (79%), FSHR was expressed by blood vessels associated with metastatic tissue. FSHR-positive vessels were present in the interior of the tumors and some few millimeters outside, in the normally appearing tissue. In the interior of the metastases, the density of the FSHR-positive vessels was constant up to 7 mm, the maximum depth available in the analyzed sections. No significant differences were noticed between the density of FSHR-positive vessels inside vs. outside tumors for metastases from lung, breast, colon, and kidney cancers. In contrast, for prostate cancer metastases, the density of FSHR-positive vessels was about 3-fold higher at the exterior of the tumor compared to the interior. Among brain metastases, the density of FSHR-positive vessels was highest in lung and kidney cancer, and lowest in prostate and colon cancer. In metastases of breast cancer to the lung pleura, the percentage of blood vessels expressing FSHR was positively correlated with the progesterone receptor level, but not with either HER-2 or estrogen receptors. In normal tissues corresponding to the host organs for the analyzed metastases, obtained from patients not known to have cancer, FSHR staining was absent, with the exception of approx. 1% of the vessels in non tumoral temporal lobe epilepsy samples. CONCLUSION: FSHR is expressed by the endothelium of blood vessels in the majority of metastatic tumors.

Escherichia coli bactofection using Lipofectamine.

Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.

Exposure to World Trade Center Dust Exacerbates Cognitive Impairment and Evokes a Central and Peripheral Pro-Inflammatory Transcriptional Profile in an Animal Model of Alzheimer's Disease.

BACKGROUND: The terrorist attacks on September 11, 2001, on the World Trade Center (WTC) led to intense fires and a massive dense cloud of toxic gases and suspended pulverized debris. In the subsequent years, following the attack and cleanup efforts, a cluster of chronic health conditions emerged among First Responders (FR) who were at Ground Zero for prolonged periods and were repeatedly exposed to high levels of WTC particulate matter (WTCPM). Among those are neurological complications which may increase the risk for the development of Alzheimer's disease (AD) later in life. OBJECTIVE: We hypothesize that WTCPM dust exposure affects the immune cross-talking between the periphery and central nervous systems that may induce brain permeability ultimately promoting AD-type phenotype. METHODS: 5XFAD and wild-type mice were intranasally administered with WTCPM dust collected at Ground Zero within 72 h after the attacks. Y-maze assay and novel object recognition behavioral tests were performed for working memory deficits and learning and recognition memory, respectively. Transcriptomic analysis in the blood and hippocampus was performed and confirmed by RT qPCR. RESULTS: Mice exposed to WTCPM dust exhibited a significant impairment in spatial and recognition short and long-term memory. Furthermore, the transcriptomic analysis in the hippocampal formation and blood revealed significant changes in genes related to immune-inflammatory responses, and blood-brain barrier disruption. CONCLUSION: These studies suggest a putative peripheral-brain immune inflammatory cross-talking that may potentiate cognitive decline, identifying for the first time key steps which may be therapeutically targetable in future studies in WTC FR.

Endothelial follicle stimulating hormone receptor in primary kidney cancer correlates with subsequent response to sunitinib.

Sunitinib is an anti-angiogenic receptor tyrosine kinase inhibitor used to treat advanced metastatic renal cell carcinoma and other types of cancer. Sutent is effective in only approximately 70% of clear cell renal cell carcinoma (CCRCC) patients, has significant adverse side effects and no method is available to predict which patients will not respond. Our purpose was to explore the possibility of introducing an effective prediction method based on a marker of the tumour vasculature, the follicle stimulating hormone receptor (FSHR). Fifty patients diagnosed with advanced metastatic CCRCC have been subjected to surgery for removal of the primary tumour and were subsequently treated with sunitinib. After three months of therapy the patients were categorized as 'responsive', 'stable' or 'non-responsive' based on the RECIST guidelines. The blood vessel density and the percentage of FSHR-positive vessels were determined by immunofluorescence on sections from the primary tumours removed by surgery, prior to the sunitinib treatment. The percentage of FSHR-stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non-responsive group. The percentage allowed the detection of responders with 87-100% sensitivity and specificity. No significant differences were detected in the total density of vessels among the three groups. The data suggest that FSHR expression levels in the blood vessels of CCRCC primary tumours can be used to predict, with high sensitivity and specificity, the patients who will respond to sunitinib therapy.

The carboxy-terminal fragment of inhibitor-2 of protein phosphatase-2A induces Alzheimer disease pathology and cognitive impairment.

Development of rational therapeutic treatments of Alzheimer disease (AD) requires the elucidation of the etiopathogenic mechanisms of neurofibrillary degeneration and ß-amyloidosis, the two hallmarks of this disease. Here we show, employing an adeno-associated virus serotype 1 (AAV1)-induced expression of the C-terminal fragment (I(2CTF)) of I(2)(PP2A), also called SET, in rat brain, decrease in protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau, and neurodegeneration; littermates treated identically but with vector only, i.e., AAV1-enhanced green fluorescent protein (GFP), served as a control. Furthermore, there was an increase in the level of activated glycogen synthase kinase-3ß and enhanced expression of intraneuronal Aß in AAV1-I(2CTF) animals. Morris water maze behavioral test revealed that infection with AAV1-I(2CTF) induced spatial reference memory and memory consolidation deficits and a decrease in the brain level of pSer133-CREB. These findings suggest a novel etiopathogenic mechanism of AD, which is initiated by the cleavage of I(2)(PP2A), producing I(2CTF), and describe a novel disease-relevant nontransgenic animal model of AD.

Microarray database mining and cell differentiation defects in schizophrenia.

The causes of schizophrenia remain unknown, but a key role of oligodendrocytes and of the myelination process carried out by them has gained increasing support. The adult human brain parenchyma contains a relatively large population of progenitor cells that can generate oligodendrocytes. Defects in these adult oligodendrocyte progenitor cells (OPCs) or in their proliferation/differentiation have received little attention as potential causes of schizophrenia yet. We compared the set of genes whose expression is modified in schizophrenia, as revealed by our microarray studies, with genes specifically expressed in stem cells, as revealed by studies on human embryonic stem cells. We also evaluated the genes that are upregulated when stem cells engage in differentiation programs. These genes can be viewed as fingerprints or signatures for differentiation processes. The comparisons revealed that a substantial fraction of the genes downregulated in the brains of persons with schizophrenia belong to the differentiation signature. A plausible interpretation of our observations is that a cell differentiation process, possibly of adult OPCs to oligodendrocytes, is perturbed in schizophrenia. These observations constitute an incentive for a new direction of study, aimed at investigating the potential role of OPCs in schizophrenia.

Pine Bark Polyphenolic Extract Attenuates Amyloid-ß and Tau Misfolding in a Model System of Alzheimer's Disease Neuropathology.

Plant-derived polyphenolic compounds possess diverse biological activities, including strong anti-oxidant, anti-inflammatory, anti-microbial, and anti-tumorigenic activities. There is a growing interest in the development of polyphenolic compounds for preventing and treating chronic and degenerative diseases, such as cardiovascular disorders, cancer, and neurological diseases including Alzheimer's disease (AD). Two neuropathological changes of AD are the appearance of neurofibrillary tangles containing tau and extracellular amyloid deposits containing amyloid-ß protein (Aß). Our laboratory and others have found that polyphenolic preparations rich in proanthocyanidins, such as grape seed extract, are capable of attenuating cognitive deterioration and reducing brain neuropathology in animal models of AD. Oligopin is a pine bark extract composed of low molecular weight proanthocyanidins oligomers (LMW-PAOs), including flavan-3-ol units such as catechin (C) and epicatechin (EC). Based on the ability of its various components to confer resilience to the onset of AD, we tested whether oligopin can specifically prevent or attenuate the progression of AD dementia preclinically. We also explored the underlying mechanism(s) through which oligopin may exert its biological activities. Oligopin inhibited oligomer formation of not only Aß1-40 and Aß1-42, but also tau in vitro. Our pharmacokinetics analysis of metabolite accumulation in vivo resulted in the identification of Me-EC-O-ß-Glucuronide, Me-(±)-C-O-ß-glucuronide, EC-O-ß-glucuronide, and (±)-C-O-ß-glucuronide in the plasma of mice. These metabolites are primarily methylated and glucuronidated C and EC conjugates. The studies conducted provide the necessary impetus to design future clinical trials with bioactive oligopin to prevent both prodromal and residual forms of AD.

The cleavage of thyroid-stimulating hormone receptor is dependent on cell-cell contacts and regulates the hormonal stimulation of phospholipase c.

Thyroid-stimulating hormone receptor (TSHR) consists of a hormone-binding extracellular subunit and a seven-transmembrane spanning subunit that interacts with the G proteins G(alphas) and G(alphaq). The two subunits, generated by proteolytic cleavage of a single polypeptide chain, are held together by disulphide bridges. The receptor is completely cleaved in thyroid tissue, while in cultured cells (thyrocytes and non-thyroid cells) the cleaved and uncleaved forms coexist. The reasons for these divergent data are not understood. Here we provide an explanation by showing that cleavage depends on cell-cell contacts. An almost complete cleavage was observed in confluent cells, while in sparse cells most of the receptor was in the uncleaved form. We also show that coupling of TSHR to G(alphaq) (as measured by inositolphosphate generation) is markedly reduced when the receptor is not cleaved. In contrast, coupling to G(alphas) [as measured by cyclic adenosine 3',5'-monophosphate (cAMP) synthesis] is unaffected by cleavage of the receptor. These results suggest that the cell-cell contacts are necessary for cleavage of the receptor, which acts as a regulatory step in inositolphosphate production via phospholipase C activation. The latter observation was confirmed using cells that express the uncleavable mutant TSHR-delta50-NET, for which the TSH-stimulated inositolphosphate production was completely abolished.

A mechanism of nucleocytoplasmic trafficking for the homeodomain protein PRH.

Proline-rich homeodomain (PRH)/hematopoietically expressed homeodomain (Hex) is a homeodomain protein that plays an important role in early embryonic patterning and hematopoiesis. PRH can act as either a tumor suppressor or an oncogene and its expression is dysregulated in certain types of lymphoid and myeloid leukemias. Aberrant exclusion of PRH from the nuclei has been associated with thyroid and breast cancers and a subset of myeloid leukemias. Accordingly, nuclear localization of PRH was found to be necessary for the inhibition of eIF4E-dependent transformation. Since PRH's nuclear-cytoplasmic localization has been associated with neoplastic transformation we sought to better understand how PRH is transported to the nuclear compartment. Here, we report an essential element that controls the mechanism of PRH nucleocytoplasmic trafficking, namely that it is imported into the nuclei by Karyopherin/Importin 7. Kap7 was identified as a binding partner for PRH in a GST-pull down from a HeLa cell protein lysate, followed by mass-spectrometry. The Kap7-PRH complex is dissociated in the presence of RanGTP, as expected for a nuclear import complex. Kap7 can bind directly to PRH in a GST-pull down assay with purified proteins, as well as mediates the transport of PRH to the nuclear compartment in a digitonin permeabilized cells assay. Finally, in vivo depletion of Kap7 dramatically reduces accumulation of PRH in the nucleus. Our data open the way for investigations of the mechanism of perturbed PRH localization in tumors and possible therapeutic interventions.

Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

PTEN induces cell cycle arrest by decreasing the level and nuclear localization of cyclin D1.

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3'-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G(1)-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G(1)- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.

Long-term culture of human gingival keratinocyte progenitor cells by down-regulation of 14-3-3 sigma.

Human gingival keratinocytes in culture stop proliferating after a limited number of passages. This limitation is associated with a gradual depletion of the stem cell compartment of the cell population. Human skin keratinocytes have a three- to five-fold higher proliferation capacity under similar culture conditions, and previous studies indicated that stable down-regulation of the 14-3-3 sigma protein in these cultures prevents stem cell differentiation and generates immortal cell lines without the effects of tumorigenic transformation, e.g., genotypic alterations. In this report, we demonstrate the creation of an immortalized human gingival keratinocyte stem cell line by stable down-regulation of the 14-3-3 sigma protein. Keratinocyte cultures were generated from human subjects ranging from 17 to 92 years of age and retrovirally transduced with a 14-3-3 sigma antisense RNA expression construct. In contrast to the control cultures, which propagated for only 2-5 passages and 25-35 cell doublings, the 14-3-3 sigma-transduced cultures propagated for 11 passages and 110 cell doublings so far. The percentage of stem cells measured by clonal analysis, which gradually decreased in the control cultures, increased to a steady level of over 90% in the 14-3-3 sigma down-regulated culture. This gingival keratinocyte stem cell line and others, which can be generated using the same procedure, have the potential to be useful for studies on stem cell differentiation, for developing gene therapy procedures that target the gingival epithelium, as well as a stable platform for testing oral hygiene products and as potential material for preprosthetic surgery.

An approach to increased polyplex gene delivery by peptides selected from a phage display library.

Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles. Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage. Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant. Bacteriophages from each of the two fractions were amplified and reincubated with the cells. Three successive rounds of selection were performed. Eighteen sequenced clones revealed 14 different sequences. Two sequences were homologous to segments of the HIV gp120 protein. For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene. The addition of the peptides led to 8-14 times increase in the expression of the reporter.