PARsylation-mediated ubiquitylation: lessons from rare hereditary disease Cherubism

Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway

Regional perturbation of gene transcription is associated with intrachromosomal rearrangements and gene fusion transcripts in high grade ovarian cancer.

Genomic rearrangements are a hallmark of cancer biology and progression, allowing cells to rapidly transform through alterations in regulatory structures, changes in expression patterns, reprogramming of signaling pathways, and creation of novel transcripts via gene fusion events. Though functional gene fusions encoding oncogenic proteins are the most dramatic outcomes of genomic rearrangements, we investigated the relationship between rearrangements evidenced by fusion transcripts and local expression changes in cancer using transcriptome data alone. 9,953 gene fusion predictions from 418 primary serious ovarian cancer tumors were analyzed, identifying depletions of gene fusion breakpoints within coding regions of fused genes as well as an N-terminal enrichment of breakpoints within fused genes. We identified 48 genes with significant fusion-associated upregulation and furthermore demonstrate that significant regional overexpression of intact genes in patient transcriptomes occurs within 1 megabase of 78 novel gene fusions that function as central markers of these regions. We reveal that cancer transcriptomes select for gene fusions that preserve protein and protein domain coding potential. The association of gene fusion transcripts with neighboring gene overexpression supports rearrangements as mechanism through which cancer cells remodel their transcriptomes and identifies a new way to utilize gene fusions as indicators of regional expression changes in diseased cells with only transcriptomic data

Intra-Articular Fms-Like Tyrosine Kinase 3 Ligand Expression Is a Driving Force in Induction and Progression of Arthritis

Background: One of the hallmarks of rheumatoid arthritis (RA) is hyperplasia and inflammation of the synovial tissue being characterized by in situ occurrence of highly differentiated leukocytes. Fms-like tyrosine kinase 3 (Flt3) has a crucial role in hematopoiesis, regulation of cell proliferation, differentiation and apoptosis. Typically, Flt3 is expressed on early myeloid and lymphoid progenitors and is activated by its soluble ligand (Flt3-L). The highly differentiated cellular pattern in the synovium of the RA patients made us hypothesize that Flt3-L, with its ability to induce proliferation and differentiation, could be of importance in induction and/or progression of arthritis. Methodology/Principal Findings: To investigate occurrence of Flt3-L in RA we have measured its levels in matched serum and synovial fluid samples from 130 patients and 107 controls. To analyse the pro-inflammatory role of Flt3-L, we continuously overexpressed this protein locally in healthy mouse joints using homologous B-cell line transfected with Flt3-L gene. Additionally, recombinant Flt3-L was instillated intra-articularly in combination with peptidoglycans, a Toll Like Receptor 2-ligand with stong arthritogenic properties. Our results show significantly higher levels of Flt3-L in the synovial fluid as compared to serum levels in RA subjects (p = 0.0001). In addition, RA synovial fluid levels of Flt-3-L were significantly higher than these obtained from synovial fluids originating from non-inflammatory joint diseases (p = 0.022). Intra-articular administration of B-cell line transfected with Flt3-L gene resulted in highly erosive arthritis while inoculation of the same B-cell line without hyperexpression of Flt3-L did not induce erosivity and only in a minority of cases caused synovial proliferation! Flt3-ligand potentiated peptidoglycan induced arthritis as compared to mice injected with peptidoglycan alone (p<0.05). Conclusions/Significance: Our findings indicate that Flt3-L is strongly expressed at the site of inflammation in human RA. It exerts both pro-inflammatory and tissue destructive properties once in the joint cavity. Owing to these properties, treatment attempts to neutralize this molecule should be considered in RA

Homeodomain-Interacting Protein Kinase (HIPK)-1 Is Required for Splenic B Cell Homeostasis and Optimal T-Independent Type 2 Humoral Response

The homeodomain-interacting protein kinase (HIPK) family is comprised of four highly related serine/threonine kinases originally identified as co-repressors for various homeodomain-containing transcription factors. The HIPKs have been shown to be involved in growth regulation and apoptosis, with numerous studies highlighting HIPK regulation of the tumor suppressor p53. In this study, we have discovered a B cell homeostatic defect in HIPK1-deficient (HIPK1−/−) mice. Lymphopoietic populations within the thymus and bone marrow of HIPK1−/− mice appeared normal based upon FACS analysis; however, the spleen exhibited a reduced number of total B cells with a significant loss of transitional-1 and follicular B cell populations. Interestingly, the marginal zone B cell population was expanded in HIPK1−/− mice, yielding an increased frequency of these cells. HIPK1−/− B cells exhibited impaired cell division in response to B cell receptor cross-linking in vitro based upon thymidine incorporation or CFSE dilution; however, the addition of CD40L rescued HIPK1−/− proliferation to wild-type levels. Despite the expanded MZ B cell population in the HIPK1−/− mice, the T-independent type 2 humoral response was impaired. These data identify HIPK1 as a novel kinase required for optimal B cell function in mice

Endonuclease increases efficiency of osteoblast isolation from murine calvariae

Bone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are isolated from neonatal calvariae through digestion with collagenase. However, the number of cells collected from one pup is not sufficient for further in vitro experiments, leading to an increase in the use of euthanized pups. We hypothesized that the viscosity of digested calvariae and digestion solution supplemented with collagenase results in cell clumping and reduction of isolated cells from bones. We simply added Benzonase, a genetically engineered endonuclease that shears all forms of DNAs/RNAs, in order to reduce nucleic acid-mediated viscosity. We found that addition of Benzonase increased the number of collected osteoblasts by three fold compared to that without Benzonase through reduction of viscosity. Additionally, Benzonase has no effect on cellular identity and function. The new osteoblast isolation protocol with Benzonase minimizes the number of neonatal pups required for an in vitro study and expands the concept that isolation of other populations of cells including osteocytes that are difficult to be purified could be modified by Benzonase

Interaction with the Phosphotyrosine Binding Domain/Phosphotyrosine Interacting Domain of SHC Is Required for the Transforming Activity of the FLT4/VEGFR3 Receptor Tyrosine Kinase

The FLT4 gene encodes two isoforms of a tyrosine kinase receptor, which belongs to the family of receptors for vascular endothelial growth factor. As the result of an alternative processing of primary mRNA transcripts, the long isoform differs from the short isoform by an additional stretch of 65 amino acid residues located at the C terminus and containing three tyrosine residues, Tyr1333, Tyr1337, and Tyr1363. Only the long isoform is endowed with a transforming capacity in fibroblasts. We show that this activity is related to the capacity of the tyrosine 1337-containing sequence to interact with the phosphotyrosine binding domain of the SHC protein. This demonstrates that a functional property of this newly described domain includes relay of mitogenic signals. In addition, it shows that the same receptor can mediate different functions through the optional binding of the phosphotyrosine binding domain and that the alternative use of this domain is sufficient to direct the signal toward different pathways

RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion

Activity of transcription factors is normally regulated through interaction with other transcription factors, chromatin remodeling proteins and transcriptional co-activators. In distinction to these well-established transcriptional controls of gene expression, we have uncovered a unique activation model of transcription factors between tyrosine kinase ABL and RUNX2, an osteoblastic master transcription factor, for cancer invasion. We show that ABL directly binds to, phosphorylates, and activates RUNX2 through its SH2 domain in a kinase activity-dependent manner and that the complex formation of these proteins is required for expression of its target gene MMP13. Additionally, we show that the RUNX2 transcriptional activity is dependent on the number of its tyrosine residues that are phosphorylated by ABL. In addition to regulation of RUNX2 activity, we show that ABL transcriptionally enhances RUNX2 expression through activation of the bone morphogenetic protein (BMP)-SMAD pathway. Lastly, we show that ABL expression in highly metastatic breast cancer MDA-MB231 cells is associated with their invasive capacity and that ABL-mediated invasion is abolished by depletion of endogenous RUNX2 or MMP13. Our genetic and biochemical evidence obtained in this study contributes to a mechanistic insight linking ABL-mediated phosphorylation and activation of RUNX2 to induction of MMP13, which underlies a fundamental invasive capacity in cancer and is different from the previously described model of transcriptional activation

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in Spheroids

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production

Survivin Loss in Thymocytes Triggers p53-mediated Growth Arrest and p53-independent Cell Death

Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4− CD8− double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre–T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development