IL‐15 regulates Bcl‐2 family members Bim and Mcl‐1 through JAK/STAT and PI3K/AKT pathways in T cells

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl‐2 proteins. Cytokines signalling through the common gamma chains such as IL‐2, IL‐7 and IL‐15 are important for T‐cell survival but how these cytokines determine the expression of Bcl‐2‐family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl‐2 proteins, pro‐apoptotic Bim and anti‐apoptotic Mcl‐1, in resting C57BL/6 mouse T cells. IL‐2, IL‐7 and IL‐15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl‐1. Bim induction by IL‐15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5‐binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL‐15 stimulation. Although it also depended on JAK/STAT‐ and PI3K signalling, Mcl‐1 regulation was independent of Mcl‐1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL‐7 effect but not the IL‐15 effect on Bcl‐2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl‐1. Later downregulation of short‐lived Mcl‐1 may induce efficient, Bim‐dependent apoptosis.     

Expression of Bcl‐2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma

Coutinho‐Camillo C M, Lourenço S V, Nishimoto I N, Kowalski L P & Soares F A
(2010) Histopathology 57 , 304–316 Expression of Bcl‐2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma Aims: To characterize the expression of proteins that inhibit (Bcl‐2, Bcl‐x, Bcl‐xL, Bcl‐2‐related protein A1, BAG‐1) or promote (Bak, Bax, Bim/Bod, Bim‐Long, Bad, Bid, PUMA) apoptosis and determine possible correlations between the expression of these proteins and clinicopathological features of oral squamous cell carcinoma (OSCC). Methods and results: Two‐hundred and twenty‐nine cases of OSCC, arranged in a tissue microarray, were immunohistochemically analysed. The results demonstrated that the absence of vascular invasion was associated with increased expression of Bak, Bax, Bcl‐xL, Bcl‐2‐related protein and PUMA. Increased expression of Bim/Bod and BAG‐1 was associated with the presence of perineural infiltration. An increase in Bid and Bim‐Long expression was associated with moderately to well‐differentiated tumours. Increased expression of the Bcl‐2‐related protein and PUMA was associated with tumours occurring in the floor of mouth and increased expression of PUMA was also associated with recurrence of the tumour. Multivariate Cox analysis demonstrated that PUMA and Bim‐Long were independent factors in prognosis of OSCC. Conclusions: Our results showed the involvement of the Bcl‐2 family of proteins in OSCC tumorigenesis and suggest that the expression of apoptotic molecules might be used as a prognostic indicator for OSCC.     

Constitutive expression of the anti‐apoptotic Bcl‐2 family member A1 in murine endothelial cells leads to transplant tolerance

Anti‐apoptotic genes, including those of the Bcl‐2 family, have been shown to have dual functionality inasmuch as they inhibit cell death but also regulate inflammation. Several anti‐apoptotic molecules have been associated with endothelial cell (EC) survival following transplantation; however, their exact role has yet to be elucidated in respect to controlling inflammation. In this study we created mice expressing murine A1 (Bfl‐1), a Bcl‐2 family member, under the control of the human intercellular adhesion molecule 2 (ICAM‐2) promoter. Constitutive expression of A1 in murine vascular ECs conferred protection from cell death induced by the proinflammatory cytokine tumour necrosis factor (TNF)‐α. Importantly, in a mouse model of heart allograft transplantation, expression of A1 in vascular endothelium increased survival in the absence of CD8⁺ T cells. Better graft outcome in mice receiving an A1 transgenic heart correlated with a reduced immune infiltration, which may be related to increased EC survival and reduced expression of adhesion molecules on ECs. In conclusion, constitutive expression of the anti‐apoptotic molecule Bfl1 (A1) in murine vascular ECs leads to prolonged allograft survival due to modifying inflammation.     

Targeting FLIP and Mcl‐1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL

The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non‐steroidal anti‐inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti‐proliferative effects and increases recombinant human tumour necrosis factor‐related apoptosis‐inducing ligand (rhTRAIL)‐induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco‐2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti‐proliferative effects of sorafenib (～four‐fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (≤ 5 mm ) and sorafenib (≤ 2.5 µm ) greatly sensitized TRAIL‐sensitive and TRAIL‐resistant colon cancer cells to rhTRAIL, much more potently than either drug combined with rhTRAIL. The increase in rhTRAIL sensitivity was due to inhibition of FLIP and Mcl‐1 protein expression following aspirin and sorafenib co‐treatment, as confirmed by knock‐down studies. Next, the clinical relevance of targeting FLIP and Mcl‐1 in colon cancer was examined. Using immunohistochemistry, we found that Mcl‐1 expression was significantly increased in colon adenoma and carcinoma patient material compared to healthy colonic epithelium, similar to the enhanced FLIP expression we recently observed in colon cancer. These results underscore the potential of combining low doses of aspirin with sorafenib to inhibit proliferation and target the anti‐apoptotic proteins FLIP and Mcl‐1 in colon cancer cells. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Angiostatic Effects of NK Cell‐Derived IFN‐γ Counteracted by Tumour Cell Bcl‐xL Expression

Anti‐apoptotic proteins that block death receptor‐mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the anti‐apoptotic protein Bcl‐x_L, known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour‐progressive activity in a murine lymphoma model. Bcl‐x_L overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl‐x_L protection of tumours from NK cell‐mediated attack did not involve protection from NK cell‐mediated cytotoxicity. Instead, Bcl‐x_L‐blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell‐secreted IFN‐γ. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.     

The cyclin‐dependent kinase inhibitor R‐roscovitine down‐regulates Mcl‐1 to override pro‐inflammatory signalling and drive neutrophil apoptosis

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non‐inflammatory phagocytosis by professional phagocytes. Recently, cyclin‐dependent kinase (CDK) inhibitors (e.g. R‐roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF‐κB and MAPK pathways and key endogenous survival proteins (typified by Mcl‐1) are involved in the regulation of neutrophil apoptosis and, in cancer‐cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R‐roscovitine over‐rides TNF‐α and LPS‐induced survival (determined by morphological examination and binding of fluorescently labelled annexin‐V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF‐κB activation (assessed by cytoplasmic IκBα proteolysis and NF‐κB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down‐regulation (at protein and mRNA level) of the survival protein Mcl‐1 but not the pro‐apoptotic bcl‐2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.     

Autophagy and Bcl‐2/BNIP3 death regulatory pathway in non‐small cell lung carcinomas

We recently showed that non‐small cell lung carcinomas (NSCLCs) are of dismal prognosis when encompassing accelerated autophagic activity. The regulation of this abnormally functioning degradation system and its association with hypoxia and apoptosis in lung carcinoma patients is unexplored. In this study we used 115 NSCLC tissues to examine the immunohistochemical expression of four distinct molecules – the major regulator of autophagy Beclin 1, the anti‐apoptotic and anti‐autophagic protein Bcl‐2, the pro‐apoptotic and pro‐autophagic protein BNIP3, and a marker of hypoxia and glucolysis, the glucose transporter Glut 1. Most cases showed reduced reactivity for Beclin 1 (62%) and Bcl‐2 (82%) proteins, almost half of our sample revealed strong BNIP3 expression (57%), whereas most of the carcinomas strongly expressed Glut 1 antigen (71%). Beclin 1 expression showed no association with survival. Bcl‐2 positivity was a marker of good prognosis (p = 0.04), whereas BNIP3 (p = 0.0004) and Glut 1 (p = 0.03) expression correlated with poor outcome in Stage I disease. Autophagic status was negatively associated with Bcl‐2 (p = 0.0006), but positively with Glut 1 expression (p = 0.001). In conclusion, the accelerated autophagic status in NSCLC is unrelated to Beclin 1 and BNIP3 expression, but does show significant association with Bcl‐2 reactivity. Furthermore, we showed important correlations between glucolysis and autophagy, guiding new pathways in future lung carcinoma research.     

Overexpression of chromatin assembly factor‐1 p60, poly(ADP‐ribose) polymerase 1 and nestin predicts metastasizing behaviour of oral cancer

Mascolo M, Ilardi G, Romano M F, Celetti A, Siano M, Romano S, Luise C, Merolla F, Rocco A, Vecchione M L, De Rosa G & Staibano S
(2012) Histopathology
Overexpression of chromatin assembly factor‐1 p60, poly(ADP‐ribose) polymerase 1 and nestin predicts metastasizing behaviour of oral cancer Aims: The natural history of oral squamous cell carcinomas (OSCCs) is variable and difficult to predict. This study aimed to assess the value of the expression of poly(ADP‐ribose) polymerase 1 (PARP‐1), chromatin assembly factor‐1 (CAF‐1)/p60 and the stem cell markers CD133, CD166, CD44, CD44v6 and nestin as markers of outcome and progression‐free survival in OSCC patients. Methods: Clinical data were collected from 66 patients (41 male and 25 female, aged 29–92 years) who underwent surgery for OSCC of the tongue, floor, lips, and palate. During follow‐up (range: 12–131 months), 14 patients experienced relapse/metastasis and/or death. The study was performed by immunohistochemistry on paraffin‐embedded tumour tissues, western blot analysis of tumour protein lysates and human cell lines, and RNA silencing assays. In addition, the human papillomavirus (HPV) status of primary tumours was evaluated by immunohistochemistry and viral subtyping. Univariate and multivariate analyses were performed to determine the correlation between these parameters and the clinical and pathological variables of the study population. Results and conclusions: We found that a PARP‐1^high/CAF‐1 p60^high/nestin^high phenotype characterized the OSCCs with the worst prognosis (all HPV‐negative). This may be of benefit in clinical management, since radio‐enhancing anti‐PARP‐1 and/or anti‐CAF‐1/p60 agents may allow radioresistance to be bypassed in the nestin‐overexpressing, metastasizing OSCC cells.     

The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma

Masir N, Jones M, Lee A M, Goff L K, Clear A J, Lister A, Marafioti T & Mason D Y
(2010) Histopathology 56 , 617–626 The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma Aims: To investigate the relationship between Bcl‐2 protein expression and cell proliferation at single‐cell level in B‐cell lymphomas using double‐labelling techniques. Methods and results: The relationship between Bcl‐2 protein expression and cell proliferation was explored in 124 cases of B‐cell lymphoma using double immunofluorescence labelling for Bcl‐2 and Ki67. In follicular lymphoma, marginal zone lymphoma and a subset of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), neoplastic cells tended to lose Bcl‐2 when they are in cell cycle. This pattern is usually maintained in both follicular lymphoma and CLL/SLL when they undergo high‐grade transformation. In mantle cell lymphoma, diffuse large B‐cell lymphoma and a subset of CLL/SLL, the inverse relationship (between Bcl‐2 and Ki67) was not observed, i.e. the proliferating cells tended to show co‐expression of Bcl‐2. Conclusions: In low‐grade lymphomas, including those that are transformed, Bcl‐2 expression is lost when cell proliferate. However, in more aggressive tumours (i.e. mantle cell and de novo diffuse large B‐cell lymphomas) the inverse Bcl‐2/Ki67 relationship was not observed. It would be of interest to explore the clinical implications in lymphoma of the presence and absence of the inverse Bcl‐2/Ki67 pattern.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

The protective properties of melatonin against aluminium‐induced neuronal injury

Aluminium (Al) toxicity is closely linked to the pathogenesis of Alzheimer's disease (AD). This experimental study investigated the neuroprotective effect of melatonin (Mel; 10 mg/kg bwt) on aluminium chloride (AlCl₃; 34 mg/kg bwt) induced neurotoxicity and oxidative stress in rats. Adult male albino Wistar rats were injected with AlCl₃ for 7 days. The effect on brain structure, lipid peroxidation (LPO), nitric oxide (NO) levels, glutathione (GSH) content, antioxidant enzymes (SOD, CAT, GPx and GR), apoptotic proteins (Bax and Bcl‐2) and an apoptotic enzyme (caspase‐3) was investigated. No apparent changes occurred following the injection of melatonin. Melatonin pretreatment of the AlCl₃‐administered rats reduced brain damage, and the tissues appeared like those of the control rats. Compared to treatment with AlCl₃, pretreatment with melatonin decreased LPO and NO levels and increased the GSH content and antioxidant enzyme activity. Moreover, melatonin increased the levels of the anti‐apoptotic protein, Bcl‐2, decreased the levels of the pro‐apoptotic protein, Bax, and inhibited caspase‐3 activity. Therefore, our results indicate that melatonin may provide therapeutic value against aluminium‐induced oxidative stress and histopathological alternations in the rat brain and that these effects may be related to anti‐apoptotic and antioxidant activities.     

Nutrient Starvation Sensitizes Human Ovarian Cancer SKOV3 Cells to BH3 Mimetic via Modulation of Mitochondrial Dynamics

The aberrant proliferation of tumor cells necessitates compensatory changes in tumor metabolic processes. Previous studies on tumor growth and metabolism have established a relationship between nutrient stress and Bcl‐2 anti‐apoptotic proteins, although the mechanisms connecting these processes remain unclear. We induced nutrient deprivation in human ovarian cancer SKOV3 cells by culturing cells in Earle's balanced salt solution (EBSS) as a starvation model. We used EBSS treatment with the BH3 domain of Bcl‐2 family proteins (BH3) mimetic ABT737, which targets Bcl‐2/Bcl‐xL, to examine mitochondrial dynamics and the interactive regulatory mechanisms between nutrition and Bcl‐2 proteins. We found that EBSS combined with ABT737 can promote SKOV3 cells to undergo apoptosis and convert tubular mitochondria into small, fragmented morphologies. Bcl‐2 family proteins participated in the regulation of mitochondrial fusion and fission through apoptosis, and the decrease of Mcl‐1 expression was the key to ABT737 sensitization. Our findings showed that nutrient stress could sensitize SKOV3 cells to ABT737 via regulation of the mitochondrial dynamic balance and interaction of Bcl‐2 family proteins. Our data suggest that nutrient starvation combined with the BH3 mimetic ABT737 could reduce the required effective dose of ABT737, and that inhibition of Bcl‐2 and Mcl‐1 together with nutrient starvation could serve as an effective strategy for the treatment of human ovarian cancer. Anat Rec, 300:326–339, 2017. © 2016 Wiley Periodicals, Inc.     

Seladin‐1 and testicular germ cell tumours: new insights into cisplatin responsiveness

The molecular basis for the exquisite sensitivity of testicular germ cell tumours of adolescents and adults (TGCTs), ie seminomas and non‐seminomatous germ cell tumours, to chemo/radiotherapy has not been fully clarified so far. It has been suggested that it may be dependent on factors involved in the regulation of apoptosis. Seladin‐1 is a multi‐functional protein involved in various biological processes, including apoptosis. The aim of our study was to assess the expression of seladin‐1 in different histological types of TGCTs, known to have varying treatment sensitivity, in order to establish whether this protein may influence cisplatin responsiveness in vitro. Seladin‐1 expression levels, both at the mRNA and at the protein level, were higher in the adjacent normal parenchyma than in the pathological counterparts. In tumoural tissues, the level of expression differed among TGCT histological types. The highest tumour‐expression level was found in teratoma, whereas the lowest was detected in seminoma, corresponding to the different chemo/and radiosensitivities of these tumour types. In common with other cancers, in TGCT‐derived cell lines seladin‐1 showed anti‐apoptotic properties through inhibition of caspase‐3 activation. We confirmed our results using a non‐seminomatous cell line model (NT2) before and after differentiation with retinoic acid. Significantly higher seladin‐1 expression was observed in the differentiated derivatives (teratoma) and an inverse relationship was found between seladin‐1 expression and the amount of cleaved caspase‐3. Seladin‐1 silencing or overexpression in this cell line supports involvement of seladin‐1 in cisplatin responsiveness. Seladin‐1 silencing was associated with greater cisplatin responsiveness demonstrated by decreased cell viability and increased expression of apoptotic markers. In contrast, overexpression of seladin‐1 was associated with a higher survival rate and a clear anti‐apoptotic effect. In conclusion, we have demonstrated for the first time an important role for seladin‐1 in the biology of TGCTs and provided new insights into cisplatin responsiveness of these tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

High expression of Mcl‐1L via the MEK‐ERK‐phospho‐STAT3 (Ser727) pathway protects melanocytes and melanoma from UVB‐induced apoptosis

Ultraviolet (UV) B is a major factor in melanomagenesis. This fact is linked to the resistance of melanocytes to UVB‐induced apoptosis. In this study, we characterized the involvement of Mcl‐1L in the regulation of UVB‐induced apoptosis in melanocytes and in melanoma cells. In melanocytes, apoptosis was not evident at 24 h after UVB irradiation. The Mcl‐1L expression increased after UVB irradiation, and the high Mcl‐1L expression continued for at least 24 h. This UVB‐dependent increase in Mcl‐1L was mediated by the MEK‐ERK‐pS‐STAT3 (STAT3 phosphorylated at Ser727) pathway. The Ser727 phosphorylation facilitated nuclear localization of STAT3. In melanoma cells, the expression levels of Mcl‐1L varied depending on the cell line. WM39 melanoma cells expressed high levels of Mcl‐1L via the MEK‐ERK‐pS‐STAT3 pathway and were resistant to UVB‐induced apoptosis without up‐regulation of Mcl‐1L. In melanocytes and in WM39 cells, transfection with Mcl‐1 siRNA promoted UVB‐induced apoptosis. Immunohistochemical studies showed that melanoma cells in in situ lesions expressed high amounts of Mcl‐1L. These results indicate that the high expression of Mcl‐1L mediated by the MEK‐ERK‐pS‐STAT3 pathway protects melanocytes and melanoma cells from UVB‐induced apoptosis.     

Differences in Bcl‐2 expression by T‐cell subsets alter their balance after in vivo irradiation to favor CD4+Bcl‐2hi NKT cells

Although it is well known that in vivo radiation depletes immune cells via the Bcl‐2 apoptotic pathway, a more nuanced analysis of the changes in the balance of immune‐cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T‐cell subsets changes after increasing single doses of total body irradiation (TBI) or after fractionated irradiation of the lymphoid tissues (TLI) of mice due to differences in radioresistance and Bcl‐2 expression of the NKT‐cell and non‐NKT subsets to favor CD4⁺Bcl‐2^hi NKT cells. Reduction of the Bcl‐2^lo mature T‐cell subsets was at least 100‐fold greater than that of the Bcl‐2^hi subsets. CD4⁺ NKT cells upregulated Bcl‐2 after TBI and TLI and developed a Th2 bias after TLI, whereas non‐NKT cells failed to do so. Our previous studies showed TLI protects against graft versus host disease in wild‐type, but not in NKT‐cell‐deficient mice. The present study shows that NKT cells have a protective function even after TBI, and these cells are tenfold more abundant after an equal dose of TLI. In conclusion, differential expression of Bcl‐2 contributes to the changes in T‐cell subsets and immune function after irradiation.     

Expression of ezrin,Bcl‐2, and Ki‐67 in chondrosarcomas

Söderström M, Palokangas T, Vahlberg T, Böhling T, Aro H, Carpen O. Expression of ezrin, Bcl‐2, and Ki‐67 in chondrosarcomas. APMIS 2010; 118: 769–76. The aim of the present study was to investigate whether the expression of ezrin, a membrane‐cytoskeleton linker and regulator of cellular signaling, is associated with clinical features of chondrosarcoma. For this purpose, we studied the expression of ezrin in 54 chondrosarcomas by immunohistochemistry and correlated the expression with other tumor characteristics, markers of proliferation, apoptosis and with clinical parameters. The intensity of ezrin staining increased with the histologic grade, and a significant positive association existed between the tumor grade and ezrin expression (p = 0.0475). In addition, there was a positive correlation between the expression of ezrin and Bcl‐2, an anti‐apoptotic protein (r = 0.83, p < 0.0001), as well as between ezrin expression and increased proliferation as measured by Ki‐67 index (r = 0.70, p < 0.0001). The positive correlation of ezrin expression with Bcl‐2 and Ki‐67 as well as with tumor grade suggests that an aggressive behavior of chondrosarcoma may be related to activation of ezrin and that ezrin inhibitors could provide a much needed adjuvant therapy in chondrosarcomas. In conclusion, our results indicate that high ezrin expression correlates with aggressive features of chondrosarcomas. Further analyses on the pathways downstream of ezrin are warranted.     

BMP‐2 Regulates the Formation of Oral Sulcus in Mouse Tongue by Altering the Balance Between TIMP‐1 and MMP‐13

The aim of this study is to investigate whether BMP‐2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP‐2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P < 0.01) by stimulating the invagination of oral sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP‐2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP‐2 siRNA induced a 60% decrease in the expression of TIMP‐1 mRNA (P < 0.05) and a drastic decline in TIMP‐1 protein was observed around the oral sulcus in the BMP‐2 siRNA treated mandibles. The recombinant BMP‐2 induced a 220% increases in the expression of TIMP‐1 mRNA and the area of the immunostaining for TIMP‐1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP‐2 than the vehicle. The BMP‐2 siRNA induced a 60% increase in the expression of MMP‐13 protein and a marked increase in the staining intensity for MMP‐13 was observed in the epithelial region of the BMP‐2 siRNA treated mandibles. The recombinant BMP‐2 induced a 70% decrease in the expression of MMP‐13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP‐2, TIMP‐1, and MMP‐13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP‐2 regulates the formation of oral sulcus by altering the balance between TIMP‐1 and MMP‐13. Anat Rec 293:1408–1415, 2010. © 2010 Wiley‐Liss, Inc.