Frank Wempe

Transcription Factor PROX1 Induces Colon Cancer Progression by Promoting the Transition from Benign to Highly Dysplastic Phenotype

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

Disruption of LTBP-4 function reduces TGF-β activation and enhances BMP-4 signaling in the lung

Disruption of latent TGF-β binding protein (LTBP)–4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-β, whereas secretion of latent TGF-β was significantly increased. Expression and secretion of TGF-β2 and -β3 increased considerably. These results suggested that TGF-β activation but not secretion would be severely impaired in LTBP-4 −/− fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)–4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the −/− fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-β1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4–mediated targeting and activation of TGF-β1 leads to enhanced BMP-4 signaling in mouse lung.

Inactivation of sestrin 2 induces TGF-β signaling and partially rescues pulmonary emphysema in a mouse model of COPD

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-β binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-β and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death.

F‐Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N‐terminal F‐box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp‐1/Cul1/F‐Box) E3‐ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C‐termini using either leucine rich repeats (LRR) or tryptophan‐aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F‐Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3‐ubiquitin ligase that targets the multifunctional regulator Y‐box binding protein 1 (YB‐1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB‐1 for proteasomal degradation, FBX33 negatively interferes with YB‐1 mediated functions. In contrast to typical FBPs, FBX33 has no C‐terminal LRR or WD40 domains and associates with YB‐1 via its N‐terminus. The present study confirms the existence of a formerly hypothetical F‐Box protein in living cells and describes one of its substrates.

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/−) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Abstract Measuring NADPH oxidase (Nox)‐derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH‐stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1‐Nox2‐Nox4‐/‐) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH‐stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH‐stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH‐stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH‐dependent signal in assays utilizing lucigenin, L‐012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR‐/‐) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR‐/‐ abolished the signal in NADPH‐stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH‐dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. Graphical abstract Figure. No Caption available. HighlightsNox activity of intact cells could not be recapitulated in membranes treated with NADPH.Proteomics of membranes show P450 reductase as source of NADPH‐dependent signal.Microsomes overexpressing Cytochrome P450 system produce a NADPH‐dependent signal.Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.Knockout of POR but not p22phox abolishes the basal and Angiotensin II‐stimulated NADPH‐dependent signal in SMC membranes.

Gene trapping identifies transiently induced survival genes during programmed cell death

BackgroundThe existence of a constitutively expressed machinery for death in individual cells has led to the notion that survival factors repress this machinery and, if such factors are unavailable, cells die by default. In many cells, however, mRNA and protein synthesis inhibitors induce apoptosis, suggesting that in some cases transcriptional activity might actually impede cell death. To identify transcriptional mechanisms that interfere with cell death and survival, we combined gene trap mutagenesis with site-specific recombination (Cre/loxP system) to isolate genes from cells undergoing apoptosis by growth factor deprivation.ResultsFrom an integration library consisting of approximately 2 × 106 unique proviral integrations obtained by infecting the interleukin-3 (IL-3)-dependent hematopoietic cell line - FLOXIL3 - with U3Cre gene trap virus, we have isolated 125 individual clones that converted to factor independence upon IL-3 withdrawal. Of 102 cellular sequences adjacent to U3Cre integration sites, 17% belonged to known genes, 11% matched single expressed sequence tags (ESTs) or full cDNAs with unknown function and 72% had no match within the public databases. Most of the known genes recovered in this analysis encoded proteins with survival functions.ConclusionsWe have shown that hematopoietic cells undergoing apoptosis after withdrawal of IL-3 activate survival genes that impede cell death. This results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus. Thus, apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals, rather than a simple death by default.

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs

Deficiency of the extracellular matrix protein latent transforming growth factor-β (TGF-β)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-β, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-β activity resulting in profibrotic processes in the lung.

Ltbp4 regulates Pdgfrβ expression via TGFβ-dependent modulation of Nrf2 transcription factor function

Latent transforming growth factor beta binding protein 4 (LTBP4) belongs to the fibrillin/LTBP family of proteins and plays an important role as a structural component of extracellular matrix (ECM) and local regulator of TGFβ signaling. We have previously reported that Ltbp4S knock out mice (Ltbp4S-/-) develop centrilobular emphysema reminiscent of late stage COPD, which could be partially rescued by inactivating the antioxidant protein Sestrin 2 (Sesn2). More recent studies showed that Sesn2 knock out mice upregulate Pdgfrβ-controlled alveolar maintenance programs that protect against cigarette smoke induced pulmonary emphysema. Based on this, we hypothesized that the emphysema of Ltbp4S-/- mice is primarily caused by defective Pdgfrβ signaling. Here we show that LTBP4 induces Pdgfrβ signaling by inhibiting the antioxidant Nrf2/Keap1 pathway in a TGFβ-dependent manner. Overall, our data identified Ltbp4 as a major player in lung remodeling and injury repair.