Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis.

OBJECTIVE: The aim of the study was to examine how mechanical unloading affects articular cartilage degeneration in the patellofemoral (PF) and tibiofemoral (TF) joints of a monosodium iodoacetate (MIA)-induced rat model of osteoarthritis (OA). DESIGN: The study involved 60 male rats. OA was induced by intra-articular injecting MIA into both knee joints. All animals were equally divided into two groups: sedentary (SE) and hindlimb unloading (HU) groups. Histopathological changes in the articular cartilage of the PF and TF joints were evaluated using the Osteoarthritis Research Society International (OARSI) score and modified Mankin score at 2 and 4 weeks after MIA injection. RESULTS: In the SE and HU groups, representative histopathological changes in OA were detected in the PF and TF joints. The OARSI and modified Mankin scores for the PF and TF joints tended to increase over time after the injection of 0.2 mg or 1.0 mg of MIA in the SE and HU groups. Both the scores for the HU group were significantly lower than those for the SE group [OARSI score: P < 0.0001 (1.0-mg injection at 4 weeks); modified Mankin score: P = 0.0116 (0.2-mg injection at 4 weeks); P = 0.0004 and < 0.0001 (1.0-mg injection at 2 and 4 weeks, respectively)]. CONCLUSION: This study revealed new histological evidence that indicates that unloading condition suppresses articular cartilage degeneration and is beneficial in many areas of basal and clinical research involving OA.

Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

OBJECTIVE: SIRT6, a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases, has been implicated as a key factor in aging-related diseases. However, the role of SIRT6 in chondrocytes has not been fully explored. The purpose of this study was to examine the role of SIRT6 in human chondrocytes by inhibiting SIRT6 in vitro. DESIGN: First, the localization of SIRT6 and proliferation cell nuclear antigen (PCNA) in human cartilages was examined by immunohistochemistry. Next, SIRT6 was depleted by RNA interference (RNAi), and the effect of SIRT6 depletion on changes in gene expression, protein levels, proliferation, and senescence in human chondrocytes was assessed. Furthermore, to detect DNA damage and telomere dysfunction, γH2AX foci and telomere dysfunction-induced foci (TIFs) were examined using immunofluorescence microscopy. The protein levels of two mediators for DNA damage induced-senescence, p16 and p21, were examined by western blotting. RESULTS: Immunohistochemical analysis showed SIRT6 was preferentially expressed in the superficial zone chondrocytes and PCNA-positive cluster-forming chondrocytes in the osteoarthritic cartilage tissue samples. Real-time PCR analysis showed that matrix metalloproteinase 1 (MMP-1) and MMP-13 mRNA were significantly increased by SIRT6 inhibition. Moreover, SIRT6 inhibition significantly reduced proliferation and increased senescence associated ß-galactosidase (SA-ß-Gal)-positive chondrocytes; it also led to increased p16 levels. Immunofluorescence microscopy showed that γH2AX foci and TIFs were increased by SIRT6 inhibition. CONCLUSION: Depletion of SIRT6 in human chondrocytes caused increased DNA damage and telomere dysfunction, and subsequent premature senescence. These findings suggest that SIRT6 plays an important role in the regulation of senescence of human chondrocytes.

A radiographic analysis of alignment of the lower extremities--initiation and progression of varus-type knee osteoarthritis.

OBJECTIVE: This study aimed to investigate alignment based on age in normal knees and alignment based on deformity in osteoarthritis (OA) knees using detailed radiographic parameters. DESIGN: Various parameters were measured from weight-bearing long leg radiographs of 1251 legs (797 normal and 454 OA knees) as a cross-sectional study. Normal knees were classified by age (young, middle aged, aged, and elderly) and symptomatic OA knees on the basis of the alignment (femorotibial angle (FTA): mild, moderate, severe and profound). The mean measurements in each group were calculated and compared within each group. RESULTS: The femoral shaft showed medially bowed curvature (femoral bowing) of approximately 2° in the young normal group, which shifted to lateral bowing with age. However, OA knees showed larger lateral bowing with OA grade, which might reduce the condylar-shaft angle and subsequently shifted the mechanical axis medially. Progression of mild to moderate OA might be associated with a decreasing condylar-shaft angle (femoral condylar orientation) and widening condylar-plateau angle (joint space narrowing) rather than decreasing tibial plateau flattering. Steeping of the tibial plateau inclination due to increasing tibial plateau shift (tibial plateau compression) rather than medial tibial bowing might be the main contributor to worsening of varus deformity in knees with severe and profound OA. CONCLUSIONS: This cross-sectional study might provide the possibility of OA initiation and progression. The lateral curvature of the femoral shaft associated with aging may contribute to the initiation of varus-type OA of the knee. These changes in the femur may be followed by secondary signs of OA progression including varus femoral condylar orientation, medial joint space narrowing, and tibial plateau compression.

Arterial stiffness is increased in young women with endometriosis.

Endometriosis is a chronic gynaecological disorder that is accompanied by inflammation and oxidative stress. Atherosclerosis has a long subclinical progression in arteries of children and young adults decades before overt clinical manifestations of the disease. In this study, we determined arterial stiffness by measuring brachial-ankle pulse wave velocity (baPWV) in women with endometriosis to assess the presence of subclinical atherosclerosis. We also measured markers of inflammation and oxidative stress in women with endometriosis. baPWV in women with endometriosis aged over 30 years was significantly higher than that in women without endometriosis aged over 30 years (p < 0.05), but not in women aged less than 30. Serum high-sensitivity C-reactive protein level in women with endometriosis was significantly higher than that in controls (p < 0.05). Young women with endometriosis show significantly increased arterial stiffness, suggesting that women with endometriosis need to be cautious of the future onset of atherosclerosis.

Site-specific changes in cytokine response to septic dose of lipopolysaccharide in ovariectomized female rats.

PURPOSE: The immune response is altered according to hormonal and metabolic status. Obesity increases the inflammatory and fever response, whereas loss of gonadal steroid decreases behavioral response to immune stress. However, the immune systems of ovariectomized animals exhibiting obesity and gonadal steroid deficiency, particularly under septic conditions, have not been fully examined. In the present study, we evaluated the ovariectomy-induced changes of central and peripheral immune responses to life-threatening septic stimulus. METHODS AND RESULTS: Ovariectomized rats showed heavier body weight and lighter uterine weight when compared with gonadally intact rats. Fever response to septic dose of lipopolysaccharide (LPS) in ovariectomized rats was less evident when compared with that in gonadally intact rats. In addition, under LPS-injected septic conditions, hypothalamic gene levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and serum protein levels of IL-1ß and TNF-α in ovariectomized rats were lower than those in gonadally intact rats. On the other hand, IL-6 levels in visceral fat under septic conditions were higher in ovariectomized rats than in gonadally intact rats. CONCLUSIONS: These findings indicate that ovariectomy-induced site-specific changes in cytokine response under septic conditions. As hypothalamic, but not peripheral, pro-inflammatory cytokines are directly involved in the fever response, the attenuation of fever response observed in ovariectomized rats may be caused by a reduction in central cytokine responses.

Effect of ultra-low-dose estradiol and dydrogesterone on arterial stiffness in postmenopausal women.

Abstract Background Ultra-low-dose estradiol is known to improve menopausal symptoms and increase bone mineral density. However, the effect of ultra-low-dose estradiol on vascular function has not been clarified. Objectives We examined the effects of ultra-low-dose estradiol on brachial-ankle pulse wave velocity (baPWV) and circulating markers of cardiovascular risk. Patients and methods Twenty-eight postmenopausal women were enrolled in this study. Fourteen women received oral estradiol (0.5 mg) and dydrogesterone (5 mg) every day for 12 months (ultra-low-dose group) as hormone replacement therapy (HRT) and 14 women as a control group did not receive HRT. The baPWV, lipid profiles, homeostasis model assessment of insulin resistance (HOMA-IR) and vascular inflammatory markers were measured. Results The baPWV level significantly decreased in the ultra-low-dose group (p = 0.037), while the baPWV level did not significantly change in the control group. HOMA-IR tended to decrease in the ultra-low-dose group (p = 0.076). Systolic blood pressure and diastolic blood pressure did not change significantly in either group. Conclusion An HRT regimen using oral ultra-low-dose estradiol and dydrogesterone has an effect on arterial stiffness and insulin resistance.

A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan in gastric carcinoma.

BACKGROUND: Cancer stem cells (CSCs) may be postulated mediators of the chemoresistance. This study aimed to determine an effective signal inhibitor with effects on the proliferation of CSCs in combination with anticancer drugs. METHODS: We used three gastric cancer cell lines and three side population (SP)-enriched CSC cell lines. We examined the combined effects of inhibitors against stemness signals, including c-Met inhibitor SU11274, and five anticancer drugs on the CSC proliferation and mRNA expression of chemoresistance-associated genes. RESULTS: The IC50 of irinotecan in SP-enriched CSC was 10.5 times higher than parent OCUM-2M cells, whereas that of oxaliplatin, taxol, gemcitabine, and 5-fluorouracil was 2.0, 2.8, 2.0, and 1.2, respectively. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines. There was a synergistic antiproliferative effect with a combination of SU11274 and SN38 in SP cells, but not other inhibitors. The SU11274 significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. The SN38 plus SU11274 group more effectively suppressed in vivo tumour growth by OCUM-2M/SP cells than either group alone. CONCLUSION: Cancer stem cells have chemoresistance to irinotecan. The c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer.

Difference in the ratio of high-molecular weight (HMW) to total adiponectin and HMW adiponectin in late post-menopausal women.

OBJECTIVE: High-molecular weight (HMW) isoform level and HMW ratio have been shown to be better predictors of insulin sensitivity and metabolic syndrome than total adiponectin level.We examined the changes in circulating levels of HMW adiponectin and ratios of HMW to total adiponectin in women during the menopausal transition. METHODS: We conducted a cross-sectional study in 217 healthy women and divided them into 4 stages: 58 women in pre-menopausal, 69 women in perimenopausal, 62 women in early post-menopausal and 28 women in late post-menopausal phase. Serum levels of total adiponectin and HMW adiponectin were measured by an enzyme-linked immunosorbent assay. RESULTS: In late post-menopausal women, HMW adiponectin level was significantly higher than that in peri-menopausal women and the HMW to total adiponectin ratio was significantly lower than that in early post-menopausal women. In peri-menopausal women, HMW adiponectin level was significantly lower than that in pre-menopausal women and HMW to total adiponectin ratio was significantly lower than the ratios in pre-menopausal and early post-menopausal women. CONCLUSION: The ratio of HMW to total adiponectin is low in late post-menopausal women, though both levels of total and HMW adiponectin were high after menopause in our cross-sectional study.

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes.

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 µm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.

An EGFR inhibitor enhances the efficacy of SN38, an active metabolite of irinotecan, in SN38-refractory gastric carcinoma cells.

BACKGROUND: Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer. METHODS: Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38 were, respectively, established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell cycle on gastric cancer cells were examined. RESULTS: Gefitinib or lapatinib showed synergistic anti-tumour effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumour growth by OCUM-2M/SN38 cells than either alone group. CONCLUSION: The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumour effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism.

Association of Alzheimer disease pathology with abnormal lipid metabolism: the Hisayama Study.

OBJECTIVE: The relationship between lipid profiles and Alzheimer disease (AD) pathology at the population level is unclear. We searched for evidence of AD-related pathologic risk of abnormal lipid metabolism. METHODS: This study included brain specimens from a series of 147 autopsies performed between 1998 and 2003 of residents in Hisayama town, Japan (76 men and 71 women), who underwent clinical examinations in 1988. Lipid profiles, such as total cholesterol (TC), triglycerides, and high-density lipoprotein cholesterol (HDLC), were measured in 1988. Low-density lipoprotein cholesterol (LDLC) was calculated using the Friedewald formula. Neuritic plaques (NPs) were assessed according to the Consortium to Establish a Registry for Alzheimer's Disease guidelines (CERAD) and neurofibrillary tangles (NFTs) were assessed according to Braak stage. Associations between each lipid profile and AD pathology were examined by analysis of covariance and logistic regression analyses. RESULTS: Adjusted means of TC, LDLC, TC/HDLC, LDLC/HDLC, and non-HDLC (defined as TC-HDLC) were significantly higher in subjects with NPs, even in sparse to moderate stages (CERAD = 1 or 2), compared to subjects without NPs in multivariate models including APOE ε4 carrier and other confounding factors. The subjects in the highest quartiles of these lipid profiles had significantly higher risks of NPs compared to subjects in the lower respective quartiles, which may suggest a threshold effect. Conversely, there was no relationship between any lipid profile and NFTs. CONCLUSION: The results of this study suggest that dyslipidemia increases the risk of plaque-type pathology.

The possibility of vertical transmission of human papillomavirus through maternal milk.

Human papillomavirus (HPV) DNA has been detected in the oral cavity of infants and breast cancer tissue, suggesting its vertical transmission through maternal milk. We determined whether HPV is detected in maternal milk and is vertically transmitted by breast-feeding. Informed consent was obtained, and maternal milk samples (n=80) were analysed for high-risk HPV DNA. In 43 women, this DNA was measured in the uterine cervix. In women with positive samples, this DNA was measured in the oral cavities of their children. The domain including HPV E6 and E7 was amplified by polymerase chain reaction using consensus primers, and HPV serotype determined by electrophoresis after restriction enzyme digestion. High-risk HPV-16 was detected in two of 80 samples (2.5%), and in these two cases, high-risk HPV was not detected in the uterine cervix or oral cavity of the child. It was concluded that the infection of HPV in maternal milk is rare (2/80); vertical transmission through maternal milk was not detected in this study (0/80). HPV infection through maternal milk may occur, but its likelihood is low.

Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice.

AIMS: This study aimed at determining whether oral administration of a probiotic strain, Lactobacillus casei strain Shirota (LcS), can improve insulin resistance, which is the underlying cause of obesity-associated metabolic abnormalities, in diet-induced obesity (DIO) mice. METHODS AND RESULTS: DIO mice were fed a high-fat diet without or with 0·05% LcS for 4 weeks and then subjected to an insulin tolerance test (ITT) or oral glucose tolerance test (OGTT). Oral administration of LcS not only accelerated the reduction in plasma glucose levels during the ITT, but also reduced the elevation of plasma glucose levels during the OGTT. In addition, plasma levels of lipopolysaccharide-binding protein (LBP), which is a marker of endotoxaemia, were augmented in the murine models of obese DIO, ob/ob, db/db and KK-A(y) and compared to those of lean mice. LcS treatment suppressed the elevation of plasma LBP levels in DIO mice, but did not affect intra-abdominal fat weight. CONCLUSIONS: LcS improves insulin resistance and glucose intolerance in DIO mice. The reduction in endotoxaemia, but not intra-abdominal fat, may contribute to the beneficial effects of LcS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that LcS has the potential to prevent obesity-associated metabolic abnormalities by improving insulin resistance.

Macrophage-dominant sialadenitis in human T-cell leukemia virus type I-associated myelopathy after living-donor liver transplantation.

A 64-year-old man who suffered from human T-cell leukemia virus type I (HTLV-I)-associated myelopathy (HAM) after living-donor liver transplantation (LDLT) for liver cirrhosis due to hepatitis C virus infection complained of xerostomia. Although exocrine function test results were positive, autoantibodies including anti-SS-A/SS-B antibodies and sialography showed negative findings. Labial salivary gland biopsy revealing infiltration of 60 counts of mononuclear cells (MNCs) in minor salivary glands led to a diagnosis of Sjögren's syndrome-like sialadenitis. Immunohistochemistry demonstrated dominant CD68 staining and major histocompatibility complex class II on the surface of infiltrating MNCs. Herein we have reported a rare condition of macrophage-dominant sialadenitis in a patient with HAM after LDLT.

Insulin resistance is associated with the pathology of Alzheimer disease: the Hisayama study.

OBJECTIVE: We examined the association between diabetes-related factors and pathology of Alzheimer disease (AD) to evaluate how diabetes affects the pathogenic process of AD. METHODS: This study included specimens from a series of 135 autopsies of residents of the town of Hisayama in Fukuoka prefecture (74 men and 61 women) performed between 1998 and 2003, who underwent a 75-g oral glucose tolerance test in clinical examinations in 1988. We measured diabetes-related factors including fasting glucose, 2-hour post-load plasma glucose, fasting insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) in 1988. Neuritic plaques (NPs) were assessed according to the Consortium to Establish a Registry for Alzheimer's Disease guidelines and neurofibrillary tangles (NFTs) were assessed according to Braak stage. The associations between each factor and AD pathology were examined by analysis of covariance and logistic regression analyses. RESULTS: Higher levels of 2-hour post-load plasma glucose, fasting insulin, and HOMA-IR were associated with increased risk for NPs after adjustment for age, sex, systolic blood pressure, total cholesterol, body mass index, habitual smoking, regular exercise, and cerebrovascular disease. However, there were no relationships between diabetes-related factors and NFTs. Regarding the effects of APOE genotype on the risk of AD pathology, the coexistence of hyperglycemia and APOE epsilon4 increased the risk for NP formation. A similar enhancement was observed for hyperinsulinemia and high HOMA-IR. CONCLUSION: The results of this study suggest that hyperinsulinemia and hyperglycemia caused by insulin resistance accelerate NP formation in combination with the effects of APOE epsilon4.

Hypoxia and RAS-signaling pathways converge on, and cooperatively downregulate, the RECK tumor-suppressor protein through microRNAs.

Cancer cells show characteristic gene expression profiles. Recent studies support the potential importance of microRNA (miRNA) expression signatures as biomarkers and therapeutic targets. The membrane-anchored protease regulator RECK is downregulated in many cancers, and forced expression of RECK in tumor cells results in decreased malignancy in animal models. RECK is also essential for mammalian development. In this study, we found that RECK is a target of at least three groups of miRNAs (miR-15b/16, miR-21 and miR-372/373); that RECK mutants lacking the target sites for these miRNA show augmented tumor/metastasis-suppressor activities; and that miR-372/373 are upregulated in response to hypoxia through HIF1alpha and TWIST1, whereas miR-21 is upregulated by RAS/ERK signaling. These data indicate that the hypoxia- and RAS-signaling pathways converge on RECK through miRNAs, cooperatively downregulating this tumor suppressor and thereby promoting malignant cell behavior.

Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats.

Recent studies have suggested that intrauterine undernutrition is closely associated with the pathogenesis of diseases after birth. Perinatal undernutrition is known to disturb the development of reproductive function and delay the onset of puberty in some species. Using a rat model, we determined the effects of prenatal undernutrition on the development of the hypothalamic kisspeptin system and evaluated whether the alteration of the kisspeptin system contributes to the delayed onset of puberty induced by prenatal undernutrition. We also evaluated the effects of prenatal undernutrition on the developmental changes in serum leptin levels because leptin was a putative positive regulator of the hypothalamic kisspeptin system. We compared the timing of vaginal opening (VO) and the developmental changes in body weight, hypothalamic Kiss1 mRNA levels, and serum leptin concentrations between offspring with prenatal undernutrition (UN offspring) and normal nutrition (NN offspring). After birth, the UN offspring showed rapid growth and had caught up to body weight of the NN offspring by postnatal day 12. After postnatal day 16, the UN offspring showed significantly lower Kiss1 mRNA levels than the NN offspring, despite their significantly higher serum leptin levels (at days 20 and 28). The timing of VO in the UN offspring was delayed compared with that in the NN offspring, and chronic central injection of kisspeptin normalized the timing of VO in the UN offspring. These results suggest that decreased hypothalamic kisspeptin action contributes to the delayed onset of puberty in prenatally undernourished female rats. Increased leptin resistance in the kisspeptin system might be involved in these alterations.

Effects of VEGFR-3 phosphorylation inhibitor on lymph node metastasis in an orthotopic diffuse-type gastric carcinoma model.

BACKGROUND: Vascular endothelial growth factor receptor-3 (VEGFR-3) signalling mediates lymphangiogenesis and lymphatic invasion; however, the effect of VEGFR-3 inhibition on the lymph node (LN) metastasis remains unclear. The aim of this study is to clarify the benefit of a VEGFR-3 inhibitor Ki23057 for LN metastasis. METHODS: Ki23057 was administered orally to gastric cancer models created by orthotopic inoculation of diffuse-type gastric cancer cells, OCUM-2MLN. The effects of Ki23057 on lymphatic vessel invasion, lymphatic vessel density, and VEGFR-3 phosphorylation were examined by immunostaining or immunoblotting. RESULTS: Ki23057 inhibited the autophosphorylation of VEGFR-3, with IC50 values of 4.3 nM in the cell-free kinase assay. Murine gastric cancer models created by the orthotopic inoculation of OCUM-2MLN cells showed the diffusely infiltrating growth and frequently developed LN metastasis. The oral administration of Ki23057 significantly (P<0.01) reduced the size of orthotopic tumours and the number of the metastatic LN in gastric cancer models. The degree of lymphatic invasion and lymphangiogenesis was significantly (P<0.05) lower in the gastric tumours treated by Ki23057. Ki23057 inhibited the phosphorylation of VEGFR-3 of lymphatic endothelial cells in gastric tumours. CONCLUSION: The inhibition of lymphangiogenesis targeting VEGFR-3 phosphorylation is a therapeutic strategy for inhibiting LN metastasis of diffuse-type gastric cancer.

The membrane-anchored metalloproteinase regulator RECK stabilizes focal adhesions and anterior-posterior polarity in fibroblasts.

Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anterior-posterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell-substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.