Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer.

Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico, protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.

The impact of cell source, culture methodology, culture location, and individual donors on gene expression profiles of bone marrow-derived and adipose-derived stromal cells.

Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

Analysis of genes regulated by the transcription factor LUMAN identifies ApoA4 as a target gene in dendritic cells.

Dendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs).

DC-STAMP knock-down deregulates cytokine production and T-cell stimulatory capacity of LPS-matured dendritic cells.

BACKGROUND: Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function. RESULTS: We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction. CONCLUSIONS: We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses.

Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice.

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters.

BACKGROUND: MicroRNAs (miRNAs) play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. RESULTS: To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs). Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs) based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. CONCLUSIONS: Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

DC-STAMP interacts with ER-resident transcription factor LUMAN which becomes activated during DC maturation.

Dendritic cells (DCs) are the professional antigen-presenting cells (APC) which efficiently prime the immune response or induce tolerance. We recently identified Dendritic Cell Specific TrAnsMembrane Protein (DC-STAMP), a novel 470 amino acid protein preferentially expressed by dendritic cells. Previously we demonstrated that DC-STAMP re-localizes towards the Golgi upon DC maturation. To identify proteins that interact with DC-STAMP, a yeast-2-hybrid analysis was performed. Here, we report a physically interacting partner of DC-STAMP in the endoplasmic reticulum (ER), called LUMAN (also known as CREB3 or LZIP). LUMAN was previously described as an ER-resident transcription factor with unknown function. It is activated in a process called regulated intramembrane proteolysis (RIP), which involves translocation to the Golgi and subsequent proteolytic cleavage. The proteolytically activated form of the protein then translocates to the nucleus. Our data indicate that DC-STAMP plays an important role in the modulation of LUMAN activation. Moreover, we demonstrate that LUMAN is endogenously expressed by DC and becomes activated by RIP upon DC maturation induced by various different stimuli. These data define LUMAN/DC-STAMP as a novel regulatory circuit in DC.

Cross-talk between human dendritic cell subsets influences expression of RNA sensors and inhibits picornavirus infection.

Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs). In this study we analyzed the expression of PRRs responding to viral RNA in human monocyte-derived DCs (moDCs) under steady-state or pro-inflammatory conditions. We found that mRNA and protein levels for most PRRs were increased under pro-inflammatory conditions, with the most pronounced increases in the RIG-like helicase (RLH) family. Additionally, freshly isolated human plasmacytoid DCs (pDCs) displayed significantly higher levels of TLR7, RIG-I, MDA5 and PKR as compared to myeloid DCs and moDCs. Finally, we demonstrate for the first time that cross-talk between TLR-matured or virus-stimulated pDCs and moDCs leads to a type I interferon-dependent antiviral state in moDCs. This antiviral state was characterized by enhanced RLH expression and protection against picornavirus infection. These findings might represent a novel mechanism by which pDCs can preserve the function and viability of myeloid DCs that are attracted to a site with ongoing infection, thereby optimizing the antiviral immune response.

Mesenchymal stem cells respond to TNF but do not produce TNF.

Previously, we demonstrated that several TLRs are expressed on cord blood-derived USSC. Stimulation of USSC with TLR agonists resulted in a marked increase of IL-6 and IL-8 production. Interestingly, TNF was undetectable after TLR stimulation, which appeared to be a result of an inactivated TNF promoter in USSC. Here, we elaborate this study by demonstrating that although USSC do not produce TNF, they are susceptible to TNF stimulation, resulting in NF-kappaB translocation and cytokine production. Additionally, we compared different stem cell sources for their ability to produce TNF. Interestingly, we found that the TNF promoter in BM-MSC is inactivated as well. Like USSC, they are able to respond to TNF stimulation, but they are not able to produce TNF, even not after LPS stimulation. This limited cytokine response in combination with the well-studied immunosuppressive properties of MSC makes these cells ideal for immune-suppressive treatment modalities such as graft-versus-host disease.

MicroRNA hsa-miR-135b regulates mineralization in osteogenic differentiation of human unrestricted somatic stem cells.

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

Functional differences between mesenchymal stem cell populations are reflected by their transcriptome.

Stem cells are widely studied to enable their use in tissue repair. However, differences in function and differentiation potential exist between distinct stem cell populations. Whether those differences are due to donor variation, cell culture, or intrinsic properties remains elusive. Therefore, we compared 3 cell lines isolated from 3 different niches using the Affymetrix Exon Array platform: the cord blood-derived neonatal unrestricted somatic stem cell (USSC), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and adult adipose tissue-derived stem cells (AdAS). While donor variation was minimal, large differences between stem cells of different origin were detected. BM-MSC and AdAS, outwardly similar, are more closely related to each other than to USSC. Interestingly, USSC expressed genes involved in the cell cycle and in neurogenesis, consistent with their reported neuronal differentiation capacity. The BM-MSC signature indicates that they are primed toward developmental processes of tissues and organs derived from the mesoderm and endoderm. Remarkably, AdAS appear to be highly enriched in immune-related genes. Together, the data suggest that the different mesenchymal stem cell types have distinct gene expression profiles, reflecting their origin and differentiation potential. Furthermore, these differences indicate a demand for effective differentiation protocols tailored to each stem cell type.

OS9 interacts with DC-STAMP and modulates its intracellular localization in response to TLR ligation.

Dendritic cell-specific transmembrane protein (DC-STAMP) has been first identified as an EST in a cDNA library of human monocyte-derived dendritic cells (DC). DC-STAMP is a multimembrane spanning protein that has been implicated in skewing haematopoietic differentiation of bone marrow cells towards the myeloid lineage, and in cell fusion during osteoclastogenesis and giant cell formation. To gain molecular insight in how DC-STAMP exerts its function, DC-STAMP interacting proteins were identified in a yeast-2-hybrid analysis. Herein, we report that amplified in osteosarcoma 9 (OS9) physically interacts with DC-STAMP, and that both proteins colocalize in the endoplasmic reticulum in various cell lines, including immature DC. OS9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. Interestingly, we now demonstrate that toll-like receptor (TLR)-induced maturation of DC leads to the translocation of DC-STAMP from the ER to the Golgi while OS9 localization is unaffected. Applying TLR-expressing CHO cells we could confirm ER-to-Golgi translocation of DC-STAMP following TLR stimulation and demonstrated that the DC-STAMP/OS9 interaction is involved in this process. Collectively, the data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering, suggesting a novel role for OS9 in myeloid differentiation and cell fusion.

Toll-like receptor triggering in cord blood mesenchymal stem cells.

Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll-like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived MSCs, the expression of TLRs and ligand-induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF-kappaB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)-6 and/or IL-8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)-alpha was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF-alpha promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up-regulation of a restricted number of pro-inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.

Serial analysis of gene expression in human keratinocytes and epidermis.

The demand for large-scale gene expression analysis tools is on the rise now that several genomes have been sequenced. One of these tools, serial analysis of gene expression (SAGE), allows the qualitative as well as quantitative analysis of a large number of genes in a defined tissue or culture model. SAGE has already been successfully used to identify differentially expressed genes in normal physiological processes and pathological conditions. This chapter focuses on the SAGE protocol and its application to cultured human keratinocytes, and on MicroSAGE, an adapted protocol that allows the use of small amounts of mRNA from isolated epidermis or a skin biopsy.

Tumor necrosis factor related apoptosis inducing ligand triggers apoptosis in dividing but not in differentiating human epidermal keratinocytes.

Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis in transformed and tumor cell lines, but usually not in other cells. Here we present a study on the effect of TRAIL on cultured keratinocytes. It is shown that differentiated and undifferentiated keratinocytes undergo apoptosis after addition of TRAIL to the medium as determined by morphologic and biochemical criteria, such as cellular shrinkage and activation of caspases. The sensitivity for TRAIL differs greatly between undifferentiated and differentiating keratinocytes, however, with undifferentiated cells being much more susceptible to apoptosis. Commitment to terminal differentiation in the absence of TRAIL does not in itself induce apoptosis. In contrast to the promyelocytic cell line HL60, internucleosomal DNA fragmentation is not observed in keratinocytes, as assessed by flow cytometric analysis and agarose gel electrophoresis. Interestingly, the prime effector of DNA fragmentation, DNA fragmentation factor of 40 kDa (DFF40), is expressed in keratinocytes, yet internucleosomal cleavage fails to occur. Our data indicate that programmed cell death during keratinocyte differentiation is distinct from receptor-mediated apoptosis in response to a death ligand.

Transcriptomics and proteomics of human skin.

The epidermis protects the organism against physical, chemical and biological challenges, and it acts as a signalling interface between the environment and the body. In order to perform these functions, the epidermal keratinocytes express a wide range of genes, several of which have been characterised previously. Recently, significant progress has been made in the large-scale analysis of keratinocyte gene expression, enabling a more profound insight into keratinocyte biology and human skin diseases. Transcriptome analysis--serial analysis of gene expression (SAGE) and microarrays--and proteome analysis have been performed on intact human epidermis and on keratinocytes cultured in model systems that mimic normal and diseased human epidermis. Here, we review the current state of large-scale gene expression analysis of human skin, with an emphasis on SAGE and complementary DNA microarrays. The merits and limitations of various approaches (transcriptomics versus proteomics) are discussed and the practical issues such as sample preparation from skin biopsies, and the use of in vitro models are briefly addressed.

A partial transcriptome of human epidermis.

Serial analysis of gene expression (SAGE) is a powerful technique for global expression profiling without prior knowledge of the genes of interest. We carried out SAGE analysis of purified keratinocytes derived from human skin biopsy specimens, resulting in a partial transcriptome of human epidermis. We identified 7645 unique SAGE tags with quantitative information from 15,131 collected SAGE tags obtained from approximately 3 x 10(6) epidermal cells. This catalog contains a large number of genes that were not previously known to be expressed by human epidermis. Comparison with the databases of all known human SAGE tags allowed us to identify a number of keratinocyte-specific tags that putatively correspond to formerly unknown genes. Surprisingly, human epidermal keratinocytes in vivo show relatively low expression levels of genes typically associated with epidermal differentiation, whereas the expression levels of housekeeping genes are considerably higher than in cultured keratinocytes. This study provides a first step toward a transcriptome of human epidermis and, as such, harbors a wealth of information to identify genes involved in skin function, and candidate genes for genetic skin disorders.

Differential gene expression in premalignant human epidermis revealed by cluster analysis of serial analysis of gene expression (SAGE) libraries.

Serial analysis of gene expression (SAGE) has been used for quantitative analysis of gene expression. We applied cluster analysis on multiple SAGE libraries derived from premalignant epidermal tissue (actinic keratosis), normal human epidermis, and cultured keratinocytes. The samples were obtained from skin biopsies without contamination by dermal tissue or blood. A total of 60,000 transcripts (tags) were analyzed. Two-way cluster analysis was applied to both the transcripts and the tissues, resulting in separation of the cultured cells from the epidermal samples, and clustering of many, presumably coregulated, genes. Two clusters of genes, strongly up-regulated in the tumor tissue compared with normal epidermis, were investigated in more detail. The differential expression of genes could be confirmed in actinic keratosis from four patients. Several of these genes have been previously associated with carcinogenesis or are likely to be important on the basis of their presumed function. Automated literature search tools show that a subgroup of these genes is coexpressed in other tissues and is part of an epidermal differentiation gene cluster on chromosome 1q21. We conclude that cluster analysis on large data sets uncovers clear partitions and correlations that could be confirmed by independent methods. We predict that these partitions will lead to biological interpretations that can be relevant for understanding the processes of carcinogenesis and tumor progression.