Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.

Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.

Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases.

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Etsl in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.

Human membrane type-2 matrix metalloproteinase is defective in cell-associated activation of progelatinase A.

Transfection of the mouse membrane type-2 matrix metalloproteinase (MT2-MMP) gene into COS-1 cells resulted in activation of progelatinase A; however, that of the human gene had no effect. Expression of human and mouse MT2-MMP chimeric proteins revealed the defect of human MT2-MMP which resides in the region between amino acid (aa) residues 155 and 271. Seven aa residues in this region were not conserved between human and mouse MT2-MMP. Substitution with the corresponding mouse residue, proline-183 to serine and glutamine-185 to aspartic acid, recovered cell-associated progelatinase A activation function. These residues are located in the insertion sequence-2 (IS-2), which was conserved in six clones of the human MT2-MMP gene from different sources, except that of proline-183 which was substituted with serine from HT1080 cells. These results indicate that human MT2-MMP is defective in cell-associated activation of progelatinase A, and this is attributed to IS-2. These findings emphasize the importance of IS-2 in MT2-MMP functionality.

Expression of metastasis-associated mts1 gene is co-induced with membrane type-1 matrix metalloproteinase (MT1-MMP) during oncogenic transformation and tubular formation of Madin Darby canine kidney (MDCK) epithelial cells.

Madin-Darby canine kidney (MDCK) epithelial cells form branching tubules in three-dimensional collagen gel in the presence of hepatocyte growth factor (HGF). Membrane type-1 matrix metalloproteinase (MT1-MMP), expression of which was induced by collagen-gel culture, was demonstrated to play an essential role in tubular formation (Y. Kadono et al. Biochem Biophys Res Commun 1988; 251: 681-7 [13]). Oncogenic transformation of MDCK cells by erbB2 and v-src induced expression of MT1-MMP, loss of cell-cell adhesion and scattered invasion into collagen gel, mRNA differential display and Northern hybridization identified metastasis-associated mts1 as one of the genes co-induced with MT1-MMP by oncogenic transformation or collagen-gel culture of MDCK cells. Expression of antisense RNA to mts1 in MDCK cells interfered with the extension of tubules into the collagen gel, however, it did not affect the morphological changes induced by HGF in culture on plastic dishes. ErbB2-transformant transfected with mts1 antisense construct, which showed unaltered morphology in culture on plastic dishes, did not scatter into collagen gel but formed aggregates. These results suggested that Mts1 contributes not only to tumor invasion but also to kidney tubulogenesis in cooperation with MT1-MMP. The coordinated action of MT1-MMP and Mts1, which is responsible for the highly invasive properties of mesenchymal cells, may be involved in epithelial tubulogenesis and invasion of malignant carcinoma cells.

Overexpression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in metastatic MDCK cells transformed by v-src.

This article discusses the transformation of epithelial Madin-Durby canine kidney (MDCK) cells with v-src induced expression of membrane-type 1-matrix metalloproteinase (MT1-MMP) and metastatic growth in nude mice (Kadono Y et al., Cancer Res 1998; 58: 2240-44). To analyze genes associated with invasive phenotype of v-src MDCK cells, mRNA differential display was performed between control and the transformed cells. A clone 12', the expression of which was clearly up-regulated in the transformed cells, encoded a protein 81% homologous to human tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Northern hybridization showed that only MT1-MMP expression was enhanced and other matrix metalloproteinases (MMPs) were undetectable or rather repressed in the transformed cells. Proteolytic activity against type I gelatin was observed in v-src MDCK cells, which was inhibited only by TIMP-2 but not by TIMP-1. MDCK cells stably transfected with the MT1-MMP gene also degraded gelatin, which was selectively inhibited by TIMP-2. These results suggest that MT1-MMP, the expression of which is induced in v-src MDCK cells, degrades extracellullar matrix by itself rather than through the activation of progelatinase A, which in turn contributes to the metastasis of the transformed cells.

Severe chest pain due to gastric anisakiasis.

We treated two cases of gastric anisakiasis presenting with severe chest pain. In both cases, there was a history of prior ingestion of raw saltwater fish. After endoscopic removal of larvae, the chest pain disappeared and never recurred. Other diseases causing chest pain were ruled out by symptoms, signs, blood tests, electrocardiography, chest radiograph, and ultrasonic examination of the heart and abdomen. Thus the chest pain was considered to be caused by gastric anisakiasis. Gastric anisakiasis should be included in the differential diagnosis of acute chest pain.

Severe hypercalcemia and polyuria in a near-drowning victim.

A 73-year-old man was admitted because of near-drowning in a hot springs bath. Transient severe hypercalcemia and polyuria were seen during the first hospital day. It seemed that the hypercalcemia was due to acute intoxication from calcium contained in the water of the spring absorbed mainly through the alveoli. To our knowledge, this is the first case of acute hypercalcemia complicating a near-drowning in a hot spring. Analysis of serum and urine electrolytes during the polyuric phase revealed saline diuresis, which was probably due to interference by the hypercalcemia of the reabsorption of sodium and free water.

Parvovirus infection and generalized edema in adults.

We describe the clinical and laboratory findings of 7 adult patients with serological evidence of recent human parvovirus B19 (HPV) infection who presented with generalized edema. Six of the 7 patients had household contact with children with erythema infectiosum and had flu-like symptoms before visiting hospital. The interval between the flu-like episode and the development of edema ranged from 4 to 13 days (mean 7.0). In all 7 patients, there was serological confirmation of recent HPV infection, and all showed the development of edema following HPV infection without urine abnormalities or anemia. Two patients presented hypocomplementemia, and two patients showed signs of congestive heart failure. HPV may be considered a causative agent of generalized edema not only in the fetus but also in adults and HPV infection should be included in the differential diagnosis of generalized edema formation.

Adult-onset Still's disease with submassive hepatic necrosis.

We present a 74-year old woman who was hospitalized because of typical spiking fever, evanescent rash, polyarthralgia, lymphadenopathy, and marked elevation of serum transaminases and lactate dehydrogenase (LDH) due to adult-onset Still's disease (AOSD) with submassive hepatic necrosis. All of the symptoms and abnormal laboratory findings were dramatically improved after treatment with prednisolone. The clinical course of this patient indicates that AOSD with severe hepatic necrosis can successfully be treated with early administration of corticosteroid, although it remains unknown whether the disease can remain in remission with no or minimal treatment.

Clinical trial of hepatitis B vaccine in the families of HBV carriers.

A hepatitis B (HB) vaccine was given to 75 subjects who were hospital workers and families of HBeAG positive carriers, and the anti-HBs response to the vaccination was studied. Subjects younger than 10 years of age were given 10 micrograms of vaccine and those 10 years or older, 20 micrograms. The second and third injections were given 4 and 24 weeks after the first. Twenty-eight weeks after the first vaccination, anti-HBs was detected in 70 of the 75 subjects (93%). The anti-HBs response was more marked in females and in younger people, but the differences between males and females and between the younger and older groups were not statistically significant. In subjects under 10 years of age, an adequate anti-HBs response was obtained with 10 micrograms of HBsAg protein. There was no clear correlation between the anti-HBs response and the HLA haplotype, because even the siblings who showed the same low anti-HBs response did not have the same HLA haplotype in common, and a difference was observed in the anti-HBs response even among siblings who had the same two HLA haplotypes.

Therapeutic hepatic vein angioplasty for Budd-Chiari syndrome.

The authors report a case of Budd-Chiari syndrome treated by percutaneous transluminal angioplasty (PTA). In this case, the occlusion of three major hepatic veins with a big collateral to the inferior vena cava via the right inferior hepatic vein (RIHV) and stenosis of the ostium of RIHV were seen. We performed successful PTA of this stenosis.

HLA antigens in Japanese patients with primary biliary cirrhosis and autoimmune hepatitis.

22 Japanese patients with primary biliary cirrhosis and 12 patients with autoimmune hepatitis were studied for HLA antigens. In the patients with primary biliary cirrhosis, HLA-DR2 showed a statistically higher frequency compared to the controls (68 versus 30%, chi 2 corr. = 7.660, p less than 0.007, p corr. less than 0.042, RR = 5.00). In the patients with autoimmune hepatitis, HLA-A10 showed a somewhat higher frequency compared to the controls (50 versus 19%, chi 2 corr. = 4.824, p less than 0.05, p corr. NS, RR = 4.36). In the DR locus, DR2 and DR4 showed tendencies toward increased frequency, but these were not statistically significant.

Familial primary biliary cirrhosis. Immunological and genetic study.

A 53-year old male and his daughter (38 years old) had primary biliary cirrhosis. The daughter had a high titer of antimitochondrial and other autoantibodies. Nine members, including, four living sibs, mother and two of the daughter's healthy offspring have been examined for liver function, the presence of autoimmune antibodies, cell mediated immunity and histocompatibility antigens. The eldest and second sons, mother and sisters of the affected daughter had multiple autoimmune reactions including antismooth-muscle antibodies and decreased peripheral T lymphocyte but liver tests were normal except in the eldest son. Histocompatibility antigens HL-A9 and HL-B5 were present in both patients and the elder son. Thus the findings suggest a similar genetic predisposition to immunologic abnormalities among the family members of the patients with primary biliary cirrhosis.