ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity.

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape.

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling.

AIMS: Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. METHODS AND RESULTS: Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E'/A'. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. CONCLUSIONS: MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Establishing normal metabolism and differentiation in hepatocellular carcinoma cells by culturing in adult human serum.

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22-32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent with in vivo hepatocytes, including the formation of large lipid and glycogen stores, increased glycogenesis, increased beta-oxidation and ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, VLDL and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur and provide insight in some of the serum components that may play a role in the establishment of the differentiated phenotype.

Inhibition of Soluble Epoxide Hydrolase Limits Mitochondrial Damage and Preserves Function Following Ischemic Injury.

AIMS: Myocardial ischemia can result in marked mitochondrial damage leading to cardiac dysfunction, as such identifying novel mechanisms to limit mitochondrial injury is important. This study investigated the hypothesis that inhibiting soluble epoxide hydrolase (sEH), responsible for converting epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids protects mitochondrial from injury caused by myocardial infarction. METHODS: sEH null and WT littermate mice were subjected to surgical occlusion of the left anterior descending (LAD) artery or sham operation. A parallel group of WT mice received an sEH inhibitor, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (tAUCB; 10 mg/L) or vehicle in the drinking water 4 days prior and 7 days post-MI. Cardiac function was assessed by echocardiography prior- and 7-days post-surgery. Heart tissues were dissected into infarct, peri-, and non-infarct regions to assess ultrastructure by electron microscopy. Complexes I, II, IV, citrate synthase, PI3K activities, and mitochondrial respiration were assessed in non-infarct regions. Isolated working hearts were used to measure the rates of glucose and palmitate oxidation. RESULTS: Echocardiography revealed that tAUCB treatment or sEH deficiency significantly improved systolic and diastolic function post-MI compared to controls. Reduced infarct expansion and less adverse cardiac remodeling were observed in tAUCB-treated and sEH null groups. EM data demonstrated mitochondrial ultrastructure damage occurred in infarct and peri-infarct regions but not in non-infarct regions. Inhibition of sEH resulted in significant improvements in mitochondrial respiration, ATP content, mitochondrial enzymatic activities and restored insulin sensitivity and PI3K activity. CONCLUSION: Inhibition or genetic deletion of sEH protects against long-term ischemia by preserving cardiac function and maintaining mitochondrial efficiency.

Matrix metalloproteinase-2 proteolysis of calponin-1 contributes to vascular hypocontractility in endotoxemic rats.

OBJECTIVE: Matrix metalloproteinase (MMP)-2 is activated in aorta during endotoxemia and plays a role in the hypocontractility to vasoconstrictors. Calponin-1 is a regulator of vascular smooth muscle tone with similarities to troponin, a cardiac myocyte protein that is cleaved by MMP-2 in myocardial oxidative stress injuries. We hypothesized that calponin-1 may be proteolyzed by MMP-2 in endotoxemia-induced vascular hypocontractility. METHODS AND RESULTS: Rats were given a nonlethal dose of bacterial lipopolysaccharide (LPS) or vehicle. Some rats were given the MMP inhibitors ONO-4817 or doxycycline. Six hours later, plasma nitrate+nitrite increased >15-fold in LPS-treated rats, an effect unchanged by doxycycline. Both ONO-4817 and doxycycline prevented LPS-induced aortic hypocontractility to phenylephrine. LPS activated MMP-2 in the aorta by S-glutathiolation. Calponin-1 levels decreased by 25% in endotoxemic aortae, which was prevented by doxycycline. Calponin-1 and MMP-2 coimmunoprecipitated and both exhibited uniform cytosolic staining in medial vascular smooth muscle cells. In vitro incubation of calponin-1 with MMP-2 led to calponin-1 degradation and appearance of its cleavage product. CONCLUSION: Calponin-1 is a target of MMP-2, which contributes to endotoxemia-induced vascular hypocontractility.

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine.

In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca (+) channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca(2+) channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

Calcium extrusion by plasma membrane calcium pump is impaired in caveolin-1 knockout mouse small intestine.

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 microM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 microM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.

Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium.

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1.

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.