Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2

TIM-1 glycoprotein mediates neutrophil peripheral recruitment during inflammation

In the last decade, the T cell immunoglobulin and mucin domain 1 (TIM-1) has emerged as a key regulator of innate and adaptive immune responses, representing a promising therapeutic target to be investigated. TIM-1 is widely distributed across immune cells, such as T, B and dendritic cells, where it exerts multiple functions. Despite being well investigated in cancer, transplantation, atopic and autoimmune diseases, several functional aspects of TIM-1 are still unclear. Recently, we have shown a novel function for TIM-1 as adhesion receptor for T helper (Th) 1 and Th17 cells, controlling their recruitment during inflammatory conditions. Neutrophils represent the first leukocyte population recruited to the injured site and several adhesion molecules are implicated in this process. Although neutrophils are classically associated with acute inflammatory processes, they are now emerging as crucial players also in chronic inflammatory diseases, including Alzheimer\u2019s disease (AD). However, the potential involvement of TIM-1 in neutrophil biology has never been investigated. The main goal of this project was to investigate neutrophil TIM-1 expression and its role in neutrophil recruitment during inflammation. By using flow cytometry and immunofluorescence staining, we demonstrated for the first time, that TIM-1 was expressed in mouse and human neutrophils, mainly stored in the cytoplasm under resting conditions. However, neutrophil activation after exposure to different inflammatory stimuli, including chemokines, fMLP, C5a, PMA and toll-like receptor (TLR) ligands rapidly triggered TIM-1 translocation to the cell surface. In the light of previous studies describing TIM-1 as an adhesion molecule and our results showing rapid translocation on neutrophil plasma membrane in response to chemotactic signals, we next tested the involvement of TIM-1 in neutrophil adhesion during inflammatory responses. By using a model of sterile thioglycolate-induced peritonitis, we demonstrated that the systemic blockade of TIM-1 or injection of exogenous neutrophils lacking a TIM- 1 functional domain, strongly reduced neutrophil accumulation in the inflamed peritoneum, indicating that TIM-1 had a role in neutrophil recruitment during acute inflammation. 4 We also found that the oligomeric form of amyloid b 1-42 (Ab1-42) peptide, a potent inflammatory mediator and pivotal key player in the development and progression of AD, strongly upregulated TIM-1 expression on neutrophil surface, suggesting a potential TIM-1 contribution in neutrophil responses during neuroinflammation. By flow cytometry, we demonstrated that TIM-1 expression increases on circulating neutrophils of 3xTg-AD mice, an animal model of AD, compared to sex- and agematched WT controls. Moreover, in 3XTg-AD mice, TIM-1-positive neutrophils accumulated in the meninges and in the choroid plexus, which are main access sites of leukocytes to the inflamed brain. Since a soluble form of TIM-1 (sTIM-1) was detected in several pathological diseases, we also measured sTIM-1 levels in the serum of 3xTg-AD mice. Interestingly, sTIM-1 significantly accumulated in the serum during disease progression, while it remained stable in the controls during aging, indicating TIM-1 as a new potential biomarker for AD. We also investigated the impact of TIM-1 on memory decline and neuropathological changes in 3xTg- AD mice crossed with TIM-1\u394mucin mice lacking functional TIM-1. By using different behavioral tests we observed a significant restoration of spatial and associative memory in 3xTg-AD/Tim-1\u394mucin mice when compared to 3xTg-AD control animals. Moreover, TIM-1 deficiency led to a drastic reduction of neutrophil accumulation in the brain during early disease and reduced neuropathological features, such as amyloid deposition, microglial activation and tau hyperphosphorylation, suggesting a role for TIM-1 in the induction of brain inflammation and neuropathological changes in an animal model of AD. Collectively, our findings shed a new light on the role of TIM-1 as a novel trafficking receptor for neutrophils suggesting that TIM-1 blockade may have a beneficial effect on the development of acute and chronic inflammatory responses

Quantitative X-ray Analysis of Montmorillonite and Kaolinite Mixtures using Zinc Oxide and Corundum Intensity Standards

Mixtures of crystalline solids can be quantified using carefully collected X-ray data and least- squares refinement of structures, in order to match calculated to experimental data. Rietveld methods are commonly found in a variety of software programs that enable crystal structure refinement. Integrated intensities of different components in a mixture are proportional to the amounts present, thus providing a powerful, nondestructive method for quantitative analysis. Layered compounds, such as clay materials in soils, often exhibit significant preferred orientation, modifying X-ray intensities and introducing challenges to quantitative analysis of samples containing clays, minerals, and amorphous components. For all samples, X-ray powder diffraction data are collected over 3-4 hours, and component structures refined. Here, we present the exploration of different techniques toward a standard procedure for soil samples. For this project, initial experiments were done with quantitative mixtures of montmorillonite or kaolinite, and goethite. After refining each component structure with Rigaku PDXL2 software, integrated intensities produced weight percents within approximately 1% (kaolinite-goethite) and 10% (montmorillonite-goethite) of lab preparations. Current experiments incorporate intensity standards by quantitative addition of zinc oxide or corundum, and these samples are being analyzed with two software programs: PDXL2 (with a FullProf software core) and the Excel-based RockJock. The former software is packaged with the diffractometer software, and the latter is freeware developed specifically for clay-containing samples. Different sample preparations to minimize the effects of preferred orientation are also being examined. All of these analyses serve the development of best practices toward gaining reliable quantitative information on clay mixtures

Peli1 impairs microglial Aβ phagocytosis through promoting C/EBPβ degradation.

Amyloid-β (Aβ) accumulation in the brain is a hallmark of Alzheimer's disease (AD) pathology. However, the molecular mechanism controlling microglial Aβ phagocytosis is poorly understood. Here we found that the E3 ubiquitin ligase Pellino 1 (Peli1) is induced in the microglia of AD-like five familial AD (5×FAD) mice, whose phagocytic efficiency for Aβ was then impaired, and therefore Peli1 depletion suppressed the Aβ deposition in the brains of 5×FAD mice. Mechanistic characterizations indicated that Peli1 directly targeted CCAAT/enhancer-binding protein (C/EBP)β, a major transcription factor responsible for the transcription of scavenger receptor CD36. Peli1 functioned as a direct E3 ubiquitin ligase of C/EBPβ and mediated its ubiquitination-induced degradation. Consequently, loss of Peli1 increased the protein levels of C/EBPβ and the expression of CD36 and thus, promoted the phagocytic ability in microglial cells. Together, our findings established Peli1 as a critical regulator of microglial phagocytosis and highlighted the therapeutic potential by targeting Peli1 for the treatment of microglia-mediated neurological diseases

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit.

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2

Wavelety seismologických signálů

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3\u2032-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation