Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

The Y14-p53 regulatory circuit in megakaryocyte differentiation and thrombocytopenia.

Thrombocytopenia-absent radius (TAR) syndrome is caused by RBM8A insufficiency. We generated megakaryocyte-specific Rbm8a knockout (Rbm8aKOMK) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, providing evidence that genetic deficiency of Rbm8a causes a disorder of platelet production. Rbm8aKOMK mice accumulated low-ploidy immature megakaryocytes in the bone marrow and exhibited defective platelet activation and aggregation. Accordingly, depletion of Y14 (RBM8A) in human erythroleukemia (HEL) cells compromised phorbol-ester-induced polyploidization. Notably, Y14/RBM8A deficiency induced both p53 and p21 in megakaryocytes and HEL cells. Treatment with a p53 inhibitor restored ex vivo differentiation of Rbm8aKOMK megakaryocytes and unexpectedly activated Y14 expression in HEL cells. Trp53 knockout partially restored megakaryocyte differentiation by reversing cell-cycle arrest and increased platelet counts of Rbm8aKOMK, indicating that excess p53 in part accounts for thrombocytopenia in TAR syndrome. This study provides evidence for the role of the Y14-p53 circuit in platelet production and a potential therapeutic strategy.

Collagenase treatment of cartilaginous matrix promotes fusion of adjacent cartilage.

In articular cartilage-repair, grafts usually fuse unsatisfactorily with surrounding host cartilage. Enzymatic dissociation of cartilaginous matrix to free chondrocytes may benefit fusion. We tested such a hypothesis with human cartilage in vitro, and with porcine cartilage in vivo. Human articular cartilage was collected from knee surgeries, cut into disc-and-ring sets, and randomly distributed into three groups: disc-and-ring sets in Group 1 were left untreated; in Group 2 only discs, and in Group 3 both discs and rings were treated with enzyme. Each disc-and-ring reassembly was cultured in a perfusion system for 14 days; expression of cartilage marker proteins and genes was evaluated by immunohistochemistry and PCR. Porcine articular cartilage from knees was similarly fashioned into disc-and-ring combinations. Specimens were randomly distributed into a control group without further treatment, and an experimental group with both disc and ring treated with enzyme. Each disc-and-ring reassembly was transplanted into subcutaneous space of a nude mouse for 30 days, and retrieved to examine disc-ring interface. In in vitro study with human cartilage, a visible gap remained at disc-ring interfaces in Group 1, yet became indiscernible in Group 2 and 3. Marker genes, including type II collagen, aggrecan and Sox 9, were well expressed by chondrocytes in all specimens, indicating that chondrocytes' phenotype retained regardless of enzymatic treatment. Similar results were found inin vivo study with porcine cartilage. Enzymatic dissociation of cartilaginous matrix promotes fusion of adjacent cartilage. The clinical relevance may be a novel method to facilitate integration of repaired cartilage in joints.

Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.

AIMS: SCUBE1 [signal peptide-CUB-epidermal growth factor (EGF) domain-containing protein 1], expressed in endothelial cells (ECs) and platelets, exists in soluble or membrane forms. We previously showed that soluble SCUBE1 is a biomarker for platelet activation and also an active participant of thrombosis. However, whether the adhesive module of its EGF-like repeats is essential and the specific contribution of SCUBE1 synthesized in ECs or platelets to thrombosis in vivo remain unclear. METHODS AND RESULTS: We generated new mutant (Δ2) mice lacking the entire EGF-like repeats to evaluate the module's functional importance during thrombogenesis in vivo. The Δ2 platelet-rich plasma showed markedly impaired platelet aggregation induced by agonists including adenosine diphosphate, collagen, the thrombin agonist PAR-4 peptide and the thromboxane A2 analogue U46619. Consistently, genetic ablation of the EGF-like repeats diminished arterial thrombosis and protected Δ2 mice against lethal thromboembolism. On flow chamber assay, whole blood isolated from Δ2 or wild-type (WT) mice pre-treated with blocking antibodies against the EGF-like repeats showed a significant decrease in platelet deposition and thrombus formation on collagen-coated surfaces under arterial shear rates. Moreover, we created animals expressing SCUBE1 only in ECs (S1-EC) or platelets (S1-PLT) by reciprocal bone-marrow transplantation between WT and Δ2 mice. The time of carotid arterial thrombosis induced by ferric chloride was normal in S1-PLT chimeric mice but much prolonged in S1-EC animals. CONCLUSIONS: We demonstrate that platelet-derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF-like repeats in vivo and suggest targeting these adhesive motifs of SCUBE1 for potential anti-thrombotic strategy.

SCUBE1-enhanced bone morphogenetic protein signaling protects against renal ischemia-reperfusion injury.

We previously reported that the membrane-bound SCUBE1 (signal peptide-CUB-epithelial growth factor domain-containing protein 1) forms a complex with bone morphogenetic protein 2 (BMP2) ligand and its receptors, thus acting as a BMP co-receptor to augment BMP signal activity. However, whether SCUBE1 can bind to and facilitate signaling activity of BMP7, a renal protective molecule for ischemia-reperfusion (I/R) insult, and contribute to the proliferation and repair of renal tubular cells after I/R remains largely unknown. In this study, we first showed that I/R-induced SCUBE1 was expressed in proximal tubular cells, which coincided with the expression of renoprotective BMP7. Molecular and biochemical analyses revealed that SCUBE1 directly binds to BMP7 and its receptors, functioning as a BMP co-receptor to promote BMP7 signaling. Furthermore, we used a new Scube1 deletion (Δ2) mouse strain to further elucidate the renal pathophysiological function of SCUBE1 after I/R injury. As compared with wild-type littermates, Δ2 mice showed severe renal histopathologic features (extensive loss of brush border, tubular necrosis, and tubular dilation) and increased inflammation (neutrophil infiltrate and induction of monocyte chemoattractant protein-1, tumor necrosis factor-α and interleukin-6) during the resolution of I/R damage. They also showed reduced BMP signaling (phosphorylated Smad1/5/8) along with decreased proliferation and increased apoptosis of renal tubular cells. Importantly, lentivirus-mediated overexpression of SCUBE1 enhanced BMP signaling and conferred a concomitant survival outcome for Δ2 proximal tubular epithelial cells after hypoxia-reoxygenation treatment. The protective BMP7 signaling may be facilitated by stress-inducible SCUBE1 after renal I/R, which suggests potential targeted approaches for acute kidney injury.

Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

OBJECTIVE: Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. APPROACH AND RESULTS: We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. CONCLUSIONS: We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy.

ATF3-mediated epigenetic regulation protects against acute kidney injury.

A variety of stress stimuli, including ischemia-reperfusion (I/R) injury, induce the transcriptional repressor ATF3 in the kidney. The functional consequences of this upregulation in ATF3 after renal I/R injury are not well understood. Here, we found that ATF3-deficient mice had higher renal I/R-induced mortality, kidney dysfunction, inflammation (number of infiltrating neutrophils, myeloperoxidase activity, and induction of IL-6 and P-selectin), and apoptosis compared with wild-type mice. Furthermore, gene transfer of ATF3 to the kidney rescued the renal I/R-induced injuries in the ATF3-deficient mice. Molecular and biochemical analysis revealed that ATF3 interacted directly with histone deacetylase 1 (HDAC1) and recruited HDAC1 into the ATF/NF-kappaB sites in the IL-6 and IL-12b gene promoters. The ATF3-associated HDAC1 deacetylated histones, which resulted in the condensation of chromatin structure, interference of NF-kappaB binding, and inhibition of inflammatory gene transcription after I/R injury. Taken together, these data demonstrate epigenetic regulation mediated by the stress-inducible gene ATF3 after renal I/R injury and suggest potential targeted approaches for acute kidney injury.

Asymmetric chitosan membrane containing collagen I nanospheres for skin tissue engineering.

A biodegradable chitosan membrane with an asymmetric structure, seeded with fibroblasts, was prepared as a novel skin substitute. Chitosan was cross-linked with genipin and then frozen and lyophilized to yield a porous asymmetric membrane (CG membrane). Nanoscale collagen I particles were injected into the CG membrane to form an asymmetric CGC membrane. The results reveal that the CG membrane treated with 0.125 wt % of genipin had a higher swelling ratio, porosity, and pore size. After 7 d of dynamic culture, many of the adhered cells exhibited a flat morphology and well spread on the surface of CGC membrane treated with 0.125 wt % of genipin. In animal studies, the CGC membrane seeded with fibroblasts and grown in vitro for 7 d was more effective than both gauze and commercial wound dressing, Suile, in healing wounds. An in vivo histological assessment indicated that covering the wound with the asymmetric CGC membrane resulted in its epithelialization and reconstruction. CGC membrane, thus, has great potential in skin tissue engineering.

JAK pathway induction of c-Myc critical to IL-5 stimulation of cell proliferation and inhibition of apoptosis.

Interleukin-5 (IL-5) induction of c-Myc expression is associated with IL-5 inhibition of apoptosis in hematopoietic cells. In this study, TFalpha1 and TFalpha8 cells with stable overexpression of IL-5 receptor alpha (IL-5Ralpha) subunit in TF-1 cells, a human hematopoietic progenitor cell line which expressed low levels of IL-5Ralpha, were used to explored how IL-5 up-regulate c-Myc and the role of c-Myc in IL-5 signaling. First, we demonstrate that IL-5 induced c-Myc RNA and protein expressions, as well as activated Janus kinases (JAK1 and JAK2) and signal transducer and activator of transcription-5b (STAT5b). JAK inhibitor AG490 and c-Myc inhibitor 10058-F4, both, reduced IL-5-mediated cell proliferation in a dose- and time-dependent manner. Both, AG490 and 10058-F4, also reduced IL-5-mediated anti-apoptotic activity. Furthermore, AG490 inhibited IL-5-mediated c-Myc induction and promoter activity. We further examined the role of JAK1 and JAK2 in the induction of c-Myc expression using the CDJAK fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1) or JAK2 (CDJAK2) as intracellular domains. Simultaneous activation of JAK1 and JAK2 by anti-CD16 antibody crosslinking of CDJAK1 and CDJAK2 could induced c-Myc expression and promoter activity; AG490 inhibited CDJAK1 and CDJAK2-mediated effects. These results suggest that IL-5 induces cell proliferation and anti-apoptosis through the JAK/c-Myc pathway, and that JAK1 and JAK2 activation participate in IL-5-induced up-regulation of c-Myc.

Octylphenol stimulates resistin gene expression in 3T3-L1 adipocytes via the estrogen receptor and extracellular signal-regulated kinase pathways.

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones. Whether environmental estrogens regulate the production of resistin is still not clear. Using 3T3-L1 adipocytes, we found that octylphenol upregulated resistin mRNA expression in dose- and time-dependent manners. The concentration of octylphenol that increased resistin mRNA levels by 50% was approximately 100 nM within 6 h of treatment. The basal half-life of resistin mRNA induced by actinomycin D was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin mRNA degradation. In addition, octylphenol stimulated resistin protein expression and release. The basal half-life of resistin protein induced by cycloheximide was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin protein degradation. While octylphenol was shown to increase activities of the estrogen receptor (ER) and MEK1, signaling was demonstrated to be blocked by pretreatment with either ICI-182780 (an ERalpha antagonist) or U-0126 (a MEK1 inhibitor), in which both inhibitors prevented octylphenol-stimulated phosphorylation of ERK. These results imply that ERalpha and ERK are necessary for the octylphenol stimulation of resistin mRNA expression. Moreover, U-0126 antagonized the octylphenol-increased resistin protein expression and release. These data suggest that the way octylphenol signaling increases resistin protein levels is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway. In vivo, octylphenol increased adipose resistin mRNA expression and serum resistin and glucose levels, supporting its in vitro effect.