Dependence of human cell survival and proliferation on the CASP3 prodomain.

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled. The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates.

Integrative analysis of KRAS wildtype metastatic pancreatic ductal adenocarcinoma reveals mutation and expression-based similarities to cholangiocarcinoma.

Oncogenic KRAS mutations are absent in approximately 10% of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) and may represent a subgroup of mPDAC with therapeutic options beyond standard-of-care cytotoxic chemotherapy. While distinct gene fusions have been implicated in KRAS wildtype mPDAC, information regarding other types of mutations remain limited, and gene expression patterns associated with KRAS wildtype mPDAC have not been reported. Here, we leverage sequencing data from the PanGen trial to perform comprehensive characterization of the molecular landscape of KRAS wildtype mPDAC and reveal increased frequency of chr1q amplification encompassing transcription factors PROX1 and NR5A2. By leveraging data from colorectal adenocarcinoma and cholangiocarcinoma samples, we highlight similarities between cholangiocarcinoma and KRAS wildtype mPDAC involving both mutation and expression-based signatures and validate these findings using an independent dataset. These data further establish KRAS wildtype mPDAC as a unique molecular entity, with therapeutic opportunities extending beyond gene fusion events.

Implementing enhanced recovery pathways: a literature review with realist synthesis.

OBJECTIVES: Enhanced Recovery Pathways (ERPs) are an increasingly popular, evidenced-based approach to surgery, designed to improve patient outcomes and reduce costs. Despite evidence demonstrating the benefits of these pathways, implementation and adherence have been inconsistent. METHODS: Using realist synthesis, this review explored the current literature surrounding the implementation of ERPs in the UK. Knowledge consolidation between authors and consulting with field experts helped to guide the search strategy. Relevant medical and social science databases were searched from 2000 to 2016, as well as a general web search. A total of 17 papers were identified, including original research, reviews, case studies and guideline documents. Full texts were analysed, cross-examined, and data extracted and synthesised. RESULTS: Several implementation strategies were identified, including the contexts in which these operated, the subsequent mechanisms of action that were triggered, and the outcome patterns they produced. Context-Mechanism-Outcome (CMO) configurations were generated, tested, and refined. These were grouped to develop two programme theories concerning ERP implementation, one related to the strategy of consulting with staff, the other with appointing a change agent to coordinate and drive the implementation process. These theories highlight instances in which implementation could be improved. CONCLUSION: Current literature in ERP research is primarily focussed on measuring patient outcomes and cost effectiveness, and as a result, important detail regarding the implementation process is often not reported or described robustly. This review not only provides recommendations for future improvements in ERP implementation, but also highlights specific areas of focus for furthering ERP implementation research.

Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts.

Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type (WT) mice. To further understand the mechanism of action, we determined the effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells appeared earlier and more extensively in the co-cultures containing calvarial cells from bgn deficient mice than WT mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH)(2)D(3). These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis.

Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors.

Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity. In this study, we have investigated the mechanism by which TGF-beta(1), TGF-beta(2) and TGF-beta(3) stimulate biglycan mRNA expression in the osteoblastic cell line MG-63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5' DNA was found to respond to TGF-beta(1) with increased transcriptional activity in a dose-dependent manner. Also TGF-beta(2) and TGF-beta(3), two structurally highly related TGF-beta isoforms stimulated biglycan transcription. A TGF-beta responsive region was identified within the first 218 bp of the human biglycan promoter upstream from the transcriptional start site, which contained several binding sites for the transcription factor Sp1. Electrophoretic mobility shift assays with nuclear extracts from MG-63 cells showed binding of both Sp1 and Sp3 to a site at -216 to -208. When the biglycan promoter construct was co-transfected with Sp1 and Sp3 expression vectors in Sp1-deficient Drosophila Schneider-2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF-beta in MG-63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first time that TGF-beta(1) stimulation of human biglycan mRNA expression relies on increased transcription of the biglycan gene, and is mediated by members of the Sp1 family of transcription factors.

Biglycan deficiency interferes with ovariectomy-induced bone loss.

UNLABELLED: Biglycan is a matrix proteoglycan with a possible role in bone turnover. In a 4-week study with sham-operated or OVX biglycan-deficient or wildtype mice, we show that biglycan-deficient mice are resistant to OVX-induced trabecular bone loss and that there is a gender difference in the response to biglycan deficiency. INTRODUCTION: Biglycan (bgn) is a small extracellular matrix proteoglycan enriched in skeletal tissues, and biglycan-deficient male mice have decreased trabecular bone mass and bone strength. The purpose of this study was to investigate the bone phenotype of the biglycan-deficient female mice and to investigate the effect of estrogen depletion by ovariectomy (OVX). MATERIALS AND METHODS: OVX or sham operations were performed on 21-week-old mice that were divided into four groups: wt sham (n = 7), wt OVX (n = 9), bgn-deficient sham (n = 10) and bgn-deficient OVX (n = 10). The mice were killed 4 weeks after surgery. Bone mass and bone turnover were analyzed by peripheral quantitative computed tomography (pQCT), biochemical markers, and histomorphometry. RESULTS AND CONCLUSIONS: In contrast to the male mice, there were only few effects of bgn deficiency on bone metabolism in female mice, showing a clear gender difference. However, when stressed by OVX, the female bgn knockout (KO) mice were resistant to the OVX-induced trabecular bone loss. The wt mice showed a decrease in trabecular bone mineral density by pQCT measurements, a decrease in trabecular bone volume (BV/TV), and an increase in mineral apposition rate. In contrast, no significant changes were detected in bgn KO mice after OVX. In addition, analysis of the bone resorption marker deoxypyridinoline showed no significant increase in the bgn KO OVX mice compared with bgn KO sham mice. Measurements of serum osteoprotegerin (OPG) and RANKL revealed increased levels of OPG and decreased levels of RANKL in the bgn KO mice compared with wt mice. In conclusion, the bgn deficiency protects against increased trabecular bone turnover and bone loss in response to estrogen depletion, supporting the concept that bgn has dual roles in bone, where it may modulate both formation and resorption ultimately influencing the bone turnover process.

Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression.

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.