Takeharu Sakamoto

Stroma-Derived Matrix Metalloproteinase (MMP)-2 Promotes Membrane Type 1-MMP–Dependent Tumor Growth in Mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.

A membrane protease regulates energy production in macrophages by activating hypoxia-inducible factor-1 via a non-proteolytic mechanism

Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism.

Cytoplasmic tail of MT1‐MMP regulates macrophage motility independently from its protease activity

Membrane type‐1 matrix metalloproteinase (MT1‐MMP) is a proinvasive protease that regulates various cellular functions as evidenced by myriad defects in different types of cells and tissues in MT1‐MMP‐deficient (MT1−/–) mice. Here we demonstrate that MT1−/– mice exhibit fewer infiltrating macrophages into sites of inflammation. MT1−/–macrophages exhibited a reduced ability to invade reconstituted basement membrane (Matrigel) and invasion by wild type (WT) macrophages was inhibited by a synthetic MMP inhibitor (BB94) to a level similar to that of MT1−/– cells. The rate of migration of MT1−/– macrophages was also low compared to that of the WT cells and re‐expression of MT1‐MMP in MT1−/– macrophages reconstituted their migratory activity. Unexpectedly, however, BB94 did not inhibit the migration of WT macrophages. The migration‐boosting activity of MT1‐MMP is retained in a mutant that lacks most of the extracellular portion including the catalytic and hemopexin‐like domains. In contrast, deletion of the cytoplasmic (CP) tail abolished the activity completely. Thus, we have demonstrated that MT1‐MMP regulates macrophages via its invasion‐promoting protease activity as well as its CP‐dependent non‐proteolytic activity to boost cell migration.

Targeting the Warburg Effect That Arises in Tumor Cells Expressing Membrane Type-1 Matrix Metalloproteinase

Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for cellular adaptation to hypoxia, although its physiological roles and activation mechanisms during normoxia have not been studied sufficiently. The Warburg effect, which is a hallmark of malignant tumors that is characterized by increased activity of aerobic glycolysis, accompanies activation of HIF-1 during normoxia. Besides tumor cells that have multiple genetic and epigenetic alterations, normal macrophages also use glycolysis for ATP production by depending upon elevated HIF-1 activity even during normoxia. We recently found that activity of factor inhibiting HIF-1 (FIH-1) is specifically suppressed in macrophages by a nonproteolytic activity of membrane type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Thus, MT1-MMP expressed in macrophages plays a significant role in regulating HIF-1 activity during normoxia. In the light of this finding, we examined here whether MT1-MMP contributes to the Warburg effect of tumor cells. All the tumor cell lines that express MT1-MMP exhibit increased glycolytic activity, and forced expression of MT1-MMP in MT1-MMP-negative tumor cells is sufficient to induce the Warburg effect. The cytoplasmic tail of MT1-MMP mediates the stimulation of aerobic glycolysis by increasing the expression of HIF-1 target genes. Specific intervention of the MT1-MMP-mediated activation of HIF-1 in tumor cells retarded tumor growth in mice. Systemic administration of a membrane-penetrating form of the cytoplasmic tail peptide in mice to inhibit HIF-1 activation competitively also exhibited a therapeutic effect on tumors.

Mint3 Enhances the Activity of Hypoxia-inducible Factor-1 (HIF-1) in Macrophages by Suppressing the Activity of Factor Inhibiting HIF-1

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor regulating cellular responses to hypoxia and is composed of α and β subunits. During normoxia, factor inhibiting HIF-1 (FIH-1) inhibits the activity of HIF-1 by preventing HIF-1α binding to p300/CBP via modification of the Asn803 residue. However, it is not known whether FIH-1 activity can be regulated in an oxygen-independent manner. In this study, we survey possible binding proteins to FIH-1 and identify Mint3/APBA3, which has been reported to bind Alzheimer β-amyloid precursor protein. Purified Mint3 binds FIH-1 and inhibits the ability of FIH-1 to modify HIF-1α in vitro. In a reporter assay, the activity of HIF-1α is suppressed because of endogenous FIH-1 in HEK293 cells, and expression of Mint3 antagonizes this suppression. Macrophages are known to depend on glycolysis for ATP production because of elevated HIF-1 activity. FIH-1 activity is suppressed in macrophages by Mint3 so as to maintain HIF-1 activity. FIH-1 forms a complex with Mint3, and these two factors co-localize within the perinuclear region. Knockdown of Mint3 expression in macrophages leads to redistribution of FIH-1 to the cytoplasm and decreases glycolysis and ATP production. Thus, Mint3 regulates the FIH-1-HIF-1 pathway, which controls ATP production in macrophages and therefore represents a potential new therapeutic target to regulate macrophage-mediated inflammation.

Establishment of an MT4-MMP-deficient mouse strain representing an efficient tracking system for MT4-MMP/MMP-17 expression in vivo using β-galactosidase

The biological functions of membrane‐type 4 matrix metalloproteinase (MT4‐MMP/MMP‐17) are poorly understood because of the lack of a sensitive system for tracking its expression in vivo. We established a mutant mouse strain (Mt4‐mmp−/–) in which Mt4‐mmp was replaced with a reporter gene encoding β‐galactosidase (LacZ). Mt4‐mmp−/– mice had normal gestations, and no apparent defects in growth, life span and fertility. Using LacZ as a marker, we were able to monitor the expression and promoter activity of Mt4‐mmp for the first time in vivo. The tissue distribution of Mt4‐mmp mRNA correlated with LacZ expression, and we showed that Mt4‐mmp is expressed primarily in cerebrum, lung, spleen, intestine and uterus. We identified LacZ‐positive neurons in the cerebrum, smooth muscle cells in the intestine and uterus, and macrophages located in the lung alveolar or intraperitoneal space. Contrary to the reported role of MT4‐MMP as a tumor necrosis factor‐α (TNF‐α) sheddase, the lipopolysaccharide (LPS)‐induced release of TNF‐α from Mt4‐mmp−/–macrophages was similar to that in wild‐type cells, and expression of Mt4‐mmp mRNA was repressed following LPS stimulation. Thus, we have established a mutant mouse strain for analyzing the physiological functions of MT4‐MMP, which also serves as a sensitive system for monitoring and tracking the expression of MT4‐MMP in vivo.

MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells

HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

ABSTRACT Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.

Identification and Characterization of Lutheran Blood Group Glycoprotein as a New Substrate of Membrane-type 1 Matrix Metalloproteinase 1 (MT1-MMP) A SYSTEMIC WHOLE CELL ANALYSIS OF MT1-MMP-ASSOCIATING PROTEINS IN A431 CELLS

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.

Deletion of the Mint3/Apba3 Gene in Mice Abrogates Macrophage Functions and Increases Resistance to Lipopolysaccharide-induced Septic Shock

Two major metabolic systems are usually used to generate ATP: oxidative phosphorylation (OXPHOS) in the mitochondria and glycolysis. Most types of cells employ OXPHOS for ATP production during normoxia but then shift energy production from OXPHOS to glycolysis when exposed to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is the master transcription factor regulating this metabolic shift. On the other hand, macrophages are unique in making use of glycolysis for ATP generation constitutively even during normoxia. We recently proposed that in macrophages, Mint3/APBA3 inhibits factor inhibiting HIF-1 (FIH-1) during normoxia, which in turn releases the suppression of HIF-1 activity by FIH-1. To demonstrate the physiological function of APBA3 in macrophages, we established Apba3−/− mice. The mutant mice presented no apparent gross phenotype but exhibited significant resistance against LPS-induced septic shock. The level of ATP in macrophages obtained from the mutant mice was reduced to 60% of the level observed in wild type cells, which in turn led to reduced ATP-dependent activities such as glycolysis, cytokine production, and motility. We also generated mutant mice with the Apba3 gene deleted specifically from cells of the myeloid lineage and confirmed that LPS-induced septic shock is mitigated significantly. Thus, we show cell type-specific regulation of energy production by APBA3 in macrophages using genetically manipulated mice. The specific function of APBA3 in macrophages might allow us to develop therapeutics to regulate aberrant macrophage function during infection and diseases.