Human mitochondrial AAA+ ATPase SKD3/CLPB assembles into nucleotide-stabilized dodecamers.

SKD3, also known as human CLPB, belongs to the AAA+ family of ATPases associated with various activities. Mutations in the SKD3/CLPB gene cause 3-methylglutaconic aciduria type VII and congenital neutropenia. SKD3 is upregulated in acute myeloid leukemia, where it contributes to anti-cancer drug resistance. SKD3 resides in the mitochondrial intermembrane space, where it forms ATP-dependent high-molecular weight complexes, but its biological function and mechanistic links to the clinical phenotypes are currently unknown. Using sedimentation equilibrium and dynamic light scattering, we show that SKD3 is monomeric at low protein concentration in the absence of nucleotides, but it forms oligomers at higher protein concentration or in the presence of adenine nucleotides. The apparent molecular weight of the nucleotide-bound SKD3 is consistent with self-association of 12 monomers. Image-class analysis and averaging from negative-stain electron microscopy (EM) of SKD3 in the ATP-bound state visualized cylinder-shaped particles with an open central channel along the cylinder axis. The dimensions of the EM-visualized particle suggest that the SKD3 dodecamer is formed by association of two hexameric rings. While hexameric structure has been often observed among AAA+ ATPases, a double-hexamer sandwich found for SKD3 appears uncommon within this protein family. A functional significance of the non-canonical structure of SKD3 remains to be determined.

Proteolytic processing of PD-L1 by ADAM proteases in breast cancer cells.

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer.

BACKGROUND: ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells. METHODS: ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown. RESULTS: We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown. CONCLUSIONS: These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.

Metalloprotease-dependent activation of EGFR modulates CD44+/CD24- populations in triple negative breast cancer cells through the MEK/ERK pathway.

PURPOSE: The CD44+/CD24- cell phenotype is enriched in triple negative breast cancers, is associated with tumor invasive properties, and serves as a cell surface marker profile of breast cancer stem-like cells. Activation of Epidermal Growth Factor Receptor (EGFR) promotes the CD44+/CD24- phenotype, but the specific signaling pathway downstream of EGFR responsible for this effect is not clear. The purpose of this study was to determine the role of the MEK/ERK pathway in the expansion of CD44+/CD24- populations in TNBC cells in response to EGFR activation. METHODS: Representative TNBC cell lines SUM159PT (claudin-low) and SUM149PT (basal) were used to evaluate cell surface expression of CD44 and CD24 by flow cytometry in response to EGFR and MEK inhibition or activation. EGFR and ERK phosphorylation levels were analyzed by Western blotting. The relationship between EGFR phosphorylation and MEK activation score in basal and claudin-low tumors from the TCGA database was examined. RESULTS: Inhibition of ERK activation with selumetinib, a MEK1/2 inhibitor, blocked EGF-induced expansion of CD44+/CD24- populations. Sustained activation of ERK by overexpression of constitutively active MEK1 was sufficient to expand CD44+/CD24- populations in cells in which EGFR activity was blocked by either erlotinib, an EGFR kinase inhibitor, or BB-94, a metalloprotease inhibitor that prevents generation of soluble EGFR ligands. In basal and claudin-low tumors from the TCGA database, there was a positive correlation between EGFR_pY1068 and MEK activation score in tumors without genomic loss of DUSP4, a negative regulator of ERK, but not in tumors harboring DUSP4 deletion. CONCLUSION: Our results demonstrate that ERK activation is a key event in EGFR-dependent regulation of CD44+/CD24- populations. Furthermore, our findings highlight the role of ligand-mediated EGFR signaling in the control of MEK/ERK pathway output in TNBC tumors without DUSP4 loss.

Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells.

Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.

ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.

BACKGROUND: ADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3'-untranslated regions (3'UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3'UTR and they may negatively regulate ADAM12-L expression. METHODS: miR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3'UTR by miRNAs was tested using an ADAM12-L 3'UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with (35)S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors. RESULTS: Transfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3'UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression. CONCLUSIONS: The ADAM12-L 3'UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers.

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7(+) MyoD(-) undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.

Biochemical characterization of the apicoplast-targeted AAA+ ATPase ClpB from Plasmodium falciparum.

ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins.

Metalloproteinase-disintegrin ADAM12 is associated with a breast tumor-initiating cell phenotype.

Members of the ADAM family of proteases have been associated with mammary tumorigenesis. Gene profiling of human breast tumors identified several intrinsic subtypes of breast cancer, which differ in terms of their basic biology, response to chemotherapy/radiation, preferential sites of metastasis, and overall patient survival. Whether or not the expression of individual ADAM proteases is linked to a particular subtype of breast cancer and whether the functions of these ADAMs are relevant to the cancer subtype have not been investigated. We analyzed several transcriptomic datasets and found that ADAM12L is specifically up-regulated in claudin-low tumors. These tumors are poorly differentiated, exhibit aggressive characteristics, have molecular signatures of epithelial-to-mesenchymal transition (EMT), and are rich in markers of breast tumor-initiating cells (BTICs). Consistently, we find that ADAM12L, but not the alternative splice variant ADAM12S, is a part of stromal, mammosphere, and EMT gene signatures, which are all associated with BTICs. In patients with estrogen receptor-negative tumors, high expression of ADAM12L, but not ADAM12S, is predictive of resistance to neoadjuvant chemotherapy. Using MCF10DCIS.com breast cancer cells, which express the endogenous ADAM12L and efficiently form mammospheres when plated at the density of single cell per well, we show that ADAM12L plays an important role in supporting mammosphere growth. We postulate that ADAM12L may serve as a novel marker and/or a novel therapeutic target in BTICs.

ADAM12 abrogation alters immune cell infiltration and improves response to checkpoint blockade therapy in the T11 murine model of triple-negative breast cancer.

Immunosuppressive tumor microenvironment (TME) impedes anti-tumor immune responses and contributes to immunotherapy resistance in triple-negative breast cancer (TNBC). ADAM12, a member of cell surface metalloproteases, is selectively upregulated in mesenchymal/claudin-low TNBCs, where its expression is largely restricted to tumor cells. The role of cancer cell-expressed ADAM12 in modulating the immune TME is not known. We show that Adam12 knockout in the T11 mouse syngeneic transplantation model of claudin-low TNBC leads to decreased numbers of tumor-infiltrating neutrophils (TINs)/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and increased numbers of tumor-infiltrating B cells and T cells. ADAM12 loss in cancer cells increases chemotaxis of B cells in vitro and this effect is eliminated by inhibition of CXCR4, a receptor for CXCL12, or anti-CXCL12 blocking antibody. Importantly, ADAM12 loss in T11 cancer cells sensitizes tumors to anti-PD1/anti-CTLA4 combination therapy, although the initial responsiveness is followed by acquired therapy resistance. Depletion of B cells in mice eliminates the improved response to immune checkpoint blockade of Adam12 knockout T11 tumors. Analysis of gene expression data for claudin-low TNBCs from the METABRIC patient cohort shows significant inverse correlations between ADAM12 and gene expression signatures of several anti-tumor immune cell populations, as well as a significant positive correlation between ADAM12 and gene expression signature of TINs/PMN-MDSCs. Collectively, these results implicate ADAM12 in immunosuppression within the TME in TNBC.

Metalloprotease-disintegrin ADAM12 expression is regulated by Notch signaling via microRNA-29.

Metalloprotease-disintegrin ADAM12 is overexpressed and frequently mutated in breast cancer. We report here that ADAM12 expression in cultured mammalian cells is up-regulated by Notch signals. Expression of a constitutively active form of Notch1 in murine fibroblasts, myoblasts, or mammary epithelial cells or activation of the endogenous Notch signaling by co-culture with ligand-expressing cells increases ADAM12 protein and mRNA levels. Up-regulation of ADAM12 expression by Notch requires new transcription, is activated in a CSL-dependent manner, and is abolished upon inhibition of IκB kinase. Expression of a constitutively active Notch1 in NIH3T3 cells increases the stability of Adam12 mRNA. We further show that the microRNA-29 family, which has a predicted conserved site in the 3'-untranslated region of mouse Adam12, plays a critical role in mediating the stimulatory effect of Notch on ADAM12 expression. In human cells, Notch up-regulates the expression of the long form, but not the short form, of ADAM12 containing a divergent 3'-untranslated mRNA region. These studies uncover a novel paradigm in Notch signaling and establish Adam12 as a Notch-related gene.

An essential role of metalloprotease-disintegrin ADAM12 in triple-negative breast cancer.

In the absence of HER2 overexpression, triple-negative breast cancers (TNBCs) rely on signaling by epidermal growth factor receptor (EGFR/ErbB1/HER1) to convey growth signals and stimulate cell proliferation. Soluble EGF-like ligands are derived from their transmembrane precursors by ADAM proteases, but the identity of the ADAM that is primarily responsible for ligand release and activation of EGFR in TNBCs is not clear. Using publicly available gene expression data for patients with lymph node-negative breast tumors who did not receive systemic treatment, we show that ADAM12L is the only ADAM with an expression level significantly associated with decreased distant metastasis-free survival times. Similar effect was not observed for patients with ER-negative non-TNBCs. There was a positive correlation between ADAM12L and HB-EGF and EGFR in TNBCs, but not in ER-negative non-TNBCs. We further demonstrate that ectopic expression of ADAM12L increased EGFR phosphorylation in a mouse intraductal xenograft model of early breast cancer. Finally, we detect strong correlation between the level of anti-ADAM12L and anti-phospho-EGFR immunostaining in human breast tumors using tissue microarrays. These studies suggest that ADAM12L is the primary protease responsible for the activation of EGFR in early stage, lymph node-negative TNBCs. Thus, our results may provide novel insight into the biology of TNBC.

The role of SnoN in transforming growth factor beta1-induced expression of metalloprotease-disintegrin ADAM12.

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.

Human CLPB forms ATP-dependent complexes in the mitochondrial intermembrane space.

Human caseinolytic peptidase B protein homolog (CLPB), also known as suppressor of potassium transport defect 3 (SKD3), is a broadly-expressed member of the family of ATPases associated with diverse cellular activities (AAA+). Mutations in the human CLPB gene cause 3-methylglutaconic aciduria type VII. CLPB is upregulated in acute myeloid leukemia (AML), where it contributes to anti-cancer drug resistance. The biological function of CLPB in human cells and mechanistic links to the clinical phenotypes are currently unknown. Herein, subcellular fractionation of human HEK-293 and BT-549 cells showed that a single 57-kDa form of CLPB was present in the mitochondria and not in the cytosolic fraction. Immunofluorescence staining of HEK-293 and BT-549 cells with anti-CLPB antibody co-localized with the mitochondrial staining using a MitoTracker dye. In purified intact mitochondria, CLPB was protected against externally added proteinase K, but it was susceptible to degradation after disruption of the outer membrane, indicating that CLPB resides in the mitochondrial intermembrane space. Overexpressed CLPB, while properly trafficked to the mitochondria, appeared to form large clusters/aggregates that were resistant to extraction with non-ionic detergents and were readily visualized by immunofluorescence microscopy. Importantly, endogenous CLPB formed high molecular weight protein complexes in an ATP-dependent manner that were detected by blue native polyacrylamide gel electrophoresis. These results demonstrate that ATP induces a structural change in CLPB and controls its ability to self-associate or form complexes with other proteins in the intermembrane space of mitochondria.

Breast cancer-associated mutations in metalloprotease disintegrin ADAM12 interfere with the intracellular trafficking and processing of the protein.

ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E and L792F. The first 2 of these mutations involve highly conserved residues in ADAM12, and our computational sequence analysis confirms that they may be cancer-related. We show that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface.

Proteomic Characterization of the Heart and Skeletal Muscle Reveals Widespread Arginine ADP-Ribosylation by the ARTC1 Ectoenzyme.

The clostridium-like ecto-ADP-ribosyltransferase ARTC1 is expressed in a highly restricted manner in skeletal muscle and heart tissue. Although ARTC1 is well studied, the identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level have been challenging and only a few ARTC1-ADP-ribosylated targets are known. Applying our recently developed mass spectrometry-based workflow to C2C12 myotubes and to skeletal muscle and heart tissues from wild-type mice, we identify hundreds of ARTC1-ADP-ribosylated proteins whose modifications are absent in the ADP-ribosylome of ARTC1-deficient mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport, and muscle function. Validation of hemopexin (HPX) as a ARTC1-target protein confirmed the functional importance of ARTC1-mediated extracellular arginine ADP-ribosylation at the systems level.

Graft vasculopathy in a Wistar rat model of heterotopic heart transplantation depending on gender matching between donors and recipients.

INTRODUCTION: Heart transplant is an accepted treatment modality in end-stage heart failure. The graft coronary artery vasculopathy is a main concern to explain the heterogeneity of the rejection process according to the gender of the donor and recipient. AIM: To assess the severity and type of mechanisms leading to failure of the graft. MATERIAL AND METHODS: Experimental allogenic heart transplantation in the abdomen was performed on Wistar rats depending on the gender of the donor and recipient (F - female; M - male) in four groups (FF, FM, MM MF). The donor heart was implanted in the abdominal cavity of the recipient. Complete time of observation was 10 weeks. Bromodeoxyuridine was administered intraperitoneally to detect proliferating cells. RESULTS: There was 42.5% graft survival in all experiments. The mean time of graft survival was 60 ±18, 54 ±29, 58 ±23 and 64 ±18 days (FF, FM, MM and MF) and no significant difference was found in graft survival time among the four experimental groups (p = 0.73). None of the heart weight changes reached statistical significance. CONCLUSIONS: The use of an animal experimental model helps to understand the mechanisms leading to graft failure and to compare the changes that occur in rats to human hearts. The gender matching affects the survival of the transplanted heart and severity of the graft vasculopathy.

Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro.

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.

ADP-ribosylation of integrin alpha7 modulates the binding of integrin alpha7beta1 to laminin.

The extracellular domain of integrin alpha7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin alpha7beta1 has not been explored. In the present study, we show that ADP-ribosylation of integrin alpha7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of alpha7beta1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa 'stalk' region of alpha7 that takes place at micromolar NAD+ concentrations increases the binding of the alpha7beta1 dimer to laminin. Increased in vitro binding of integrin alpha7beta1 to laminin after ADP-ribosylation of the 37-kDa fragment of alpha7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of alpha7 that occurs at approx. 100 microM NAD+ inhibits the binding of integrin alpha7beta1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin beta1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin alpha7beta1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

Phenotypic diversity of breast cancer-related mutations in metalloproteinase-disintegrin ADAM12.

Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity--R612Q and L792F--occur in triple-negative breast cancers, while loss-of-function mutations--D301H, G479E, T596A, and G668A--are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.