ALK1 expression in oral lichen planus: a possible relation to microvessel density.

OBJECTIVE: To assess the expression of activin receptor-like kinase 1 (ALK1) and investigate its possible relationship with microvessel density (MVD) in different forms of oral lichen planus (OLP) compared to controls' biopsies. METHODS: Biopsies from 20 reticular/papular OLP (R/PLP), 20 atrophic/erosive OLP (A/ELP) patients, and 20 healthy subjects were immunohistochemically analyzed and statistically compared and correlated for ALK1 expression and MVD as assessed by CD34 expression. RESULTS: All OLP specimens revealed the presence of positive cytoplasmic CD34 immunostaining in endothelial cells, with statistically high significant MVD in each of R/PLP (Median; M = 4.40) and A/ELP (M = 7.69) compared to controls (M = 1.16) (P < 0.001). Statistically significant MVD was found in A/ELP compared to R/PLP (P < 0.001). All control specimens revealed negative ALK1 immunostaining of the few inflammatory cells found, while 85% of A/ELP cases and 70% of R/PLP cases showed positively immunostained sections for ALK-1, with statistically significant higher ALK1 expression In A/ELP (M = 1.95) compared to R/PLP (M = 0.86) (P = 0.005). No significant correlation between CD34 and ALK1 was detected in R/PLP (r = 0.081), while a barely moderate positive correlation was found in A/ELP (r = 0.396). CONCLUSIONS: ALK1 expression and MVD are increased in OLP, particularly in A/ELP type.

Exercise restores decreased physical activity levels and increases markers of autophagy and oxidative capacity in myostatin/activin-blocked mdx mice.

The importance of adequate levels of muscle size and function and physical activity is widely recognized. Myostatin/activin blocking increases skeletal muscle mass but may decrease muscle oxidative capacity and can thus be hypothesized to affect voluntary physical activity. Soluble activin receptor IIB (sActRIIB-Fc) was produced to block myostatin/activins. Modestly dystrophic mdx mice were injected with sActRIIB-Fc or PBS with or without voluntary wheel running exercise for 7 wk. Healthy mice served as controls. Running for 7 wk attenuated the sActRIIB-Fc-induced increase in body mass by decreasing fat mass. Running also enhanced/restored the markers of muscle oxidative capacity and autophagy in mdx mice to or above the levels of healthy mice. Voluntary running activity was decreased by sActRIIB-Fc during the first 3-4 wk correlating with increased body mass. Home cage physical activity of mice, quantified from the force plate signal, was decreased by sActRIIB-Fc the whole 7-wk treatment in sedentary mice. To understand what happens during the first weeks after sActRIIB-Fc administration, when mice are less active, healthy mice were injected with sActRIIB-Fc or PBS for 2 wk. During the sActRIIB-Fc-induced rapid 2-wk muscle growth period, oxidative capacity and autophagy were reduced, which may possibly explain the decreased running activity. These results show that increased muscle size and decreased markers of oxidative capacity and autophagy during the first weeks of myostatin/activin blocking are associated with decreased voluntary activity levels. Voluntary exercise in dystrophic mice enhances the markers of oxidative capacity and autophagy to or above the levels of healthy mice.

MicroRNA control of Nodal signalling.

MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades.

miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms.

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-ß pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-ß pathway in this process.

Expression of the TGF-beta1 system in human testicular pathologies.

BACKGROUND: In non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO) and hypospermatogenesis (H) are commonly found. In these pathologies, Leydig cell hyperplasia (LCH) is detected in some patients. Since TGF-ß1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-ß1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), and the co-receptor endoglin in human biopsies from patients with idiopathic infertility. METHODS: Specific immunostaining of TGF-ß1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-ß1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR. RESULTS: Immunohistochemical studies revealed that TGF-ß1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-ß1 signaling, are also detected in both their phosphorylated (activated) and dephosphorylated form in all samples TGF-ß1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed. CONCLUSIONS: In conclusion, the high levels of mRNA and protein expression of the TGF-ß1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.

ALK-1-positive inflammatory myofibroblastic tumor of the thyroid complicated by Hashimoto's thyroiditis: report of a rare case and a literature review.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) of the thyroid are extremely rare soft-tissue tumors. In the literature, IMTs are sometimes called plasma cell granulomas (PCGs) or inflammatory pseudotumors, which often causes ambiguity. To date, 17 cases of PCGs and five cases of thyroid IMTs have been reported. These cases reveal that IMTs of the thyroid are often negative for the anaplastic lymphoma kinase (ALK-1) gene. To provide further information on this rare lesion, we present a case of an ALK-1-positive thyroid IMT and a review of IMTs of the thyroid. CASE PRESENTATION: A 34-year-old Chinese woman presented with a painless neck mass that had persisted for over a month. Ultrasonography revealed a 4.28 × 2.53 cm2 hypoechoic mass, in the left lobe of the thyroid gland. Serum levels of thyroglobulin and anti-thyroglobulin antibodies were high. Subsequently, left lobectomy was performed. Macroscopically, the lesion was a gray-brown nodular mass with a partial envelope. Histologically, two different lesion types were observed. The first lesion showed classic spindle cell proliferation, with spindle cells arranged in fascicles, accompanied by mature inflammatory cells. The other lesion showed a large number of infiltrating lymphocytes, with lymphoid follicles in the remaining thyroid gland, which was atrophic. Immunohistochemical staining showed that the spindle cells were negative for CK19, CyclinD1, Gelectin-3, EMA, CD34, S100, Bcl-2, and STAT-6, but strongly positive for ALK-1, vimentin, and TTF1. CK was focally expressed, and the Ki-67 index was 5%. A diagnosis of IMT was proposed according to immunohistochemical findings and morphology. Hashimoto's thyroiditis was confirmed according to serum levels of thyroglobulin and anti-thyroglobulin antibodies and morphology. The patient did not receive adjuvant therapy. She remained alive without disease recurrence for 10 months after lobectomy. CONCLUSIONS: IMTs should be considered in the diagnosis when spindle cell proliferation accompanied by mature inflammatory cells is observed, spindle cells are mildly atypical, and myofibroblast differentiation is present in the thyroid. A uniform diagnostic term is crucial to avoid ambiguity. Clinicians and pathologists should be aware of the necessity for long-term follow-up, especially in ALK-positive cases. The therapeutic potential of ALK-1 positivity should be explored further.

The expression of activin-betaA- and -betaB-subunits, follistatin, and activin type II receptors in fallopian tubes bearing an ectopic pregnancy.

CONTEXT: Ectopic pregnancy is a major cause of maternal morbidity and mortality with increasing incidence worldwide. OBJECTIVE: We investigated whether epithelia from Fallopian tubes (FTs) bearing an ectopic pregnancy differ from normal tubes in expression of TGF-beta family and related proteins and their receptors. METHODOLOGY: Because it is not possible to collect FTs from women carrying a healthy pregnancy, we studied tissue collected at the time of hysterectomy for benign disease. Women were injected with human chorionic gonadotropin in the days leading up to hysterectomy to produce a state of pseudopregnancy. Pseudopregnancy status was confirmed by the presence of high serum progesterone levels and the decidualization of the endometrium. Fifteen FTs bearing ectopic pregnancy and six pseudopregnant tubes were collected and examined using immunohistochemistry and quantitative RT-PCR. RESULTS: Immunohistochemistry demonstrated clear staining for the betaA- and betaB-subunits, type II receptor group comprising the activin type IIA and type IIB receptors, and follistatin, which increased in intensity from the isthmus to the ampulla in both models. However, the intensity of expression of these molecules was stronger in the ectopic pregnancy group when compared with the pseudopregnant group. Quantitative RT-PCR showed significant decrease in mRNA levels of betaA-subunit, activin type IIA and IIB receptors, and follistatin in ectopic group (P < 0.05) but no changes in betaB-subunit (P > 0.05). Overall, there was an apparent paradox of high concentration of protein but low mRNA expression. CONCLUSION: Activin-A may stimulate tubal decidualization and trophoblast invasion. A better understanding of the mechanism by which an embryo implants in the tubal epithelium may lead to improved methods for early diagnosis and/or management of ectopic pregnancy.

Myostatin inhibits differentiation of bovine preadipocyte.

We investigated the effect of myostatin on the differentiation of bovine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with myostatin during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. The treatment with myostatin throughout the differentiation period severely suppressed the induction of all differentiation markers. The treatment with myostatin in the early phase of differentiation also suppressed the induction of terminal differentiation markers but three-fold higher dose of myostatin was required for the suppression compared with its treatment throughout the differentiation period. Myostatin treatment reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma mRNA and interfered with the induction of CCAAT/enhancer binding protein (C/EBP) alpha mRNA. We also observed that follistatin stimulates preadipocyte differentiation in the presence of myostatin. These results suggest that myostatin inhibits bovine preadiopocyte differentiation through suppressing PPARgamma and C/EBPalpha mRNA expressions and that follistatin counteracts the suppressive effect of myostatin.

Downstream mRNA Target Analysis in Neonatal Hypoxic-Ischaemic Encephalopathy Identifies Novel Marker of Severe Injury: a Proof of Concept Paper.

Human microRNA miR-374a is downregulated in the umbilical cord blood (UCB) of infants with hypoxic-ischaemic encephalopathy (HIE). The downstream targets of this microRNA (miRNA) are unclear, but one putative target is the activin-A receptor type IIb (ACVR2B). ACVR2B is required for activin-A function and previous reports have shown alterations of activin-A levels in neonatal HIE. Our aim was to investigate the expression of the potential downstream targets of miR-374a, activin-A and ACVR2B, at birth in a cohort of full-term infants with perinatal asphyxia (PA) only, and those with PA who developed clinical and electrographic HIE. UCB was drawn and processed immediately after delivery. Levels of serum activin-A were measured using ELISA. mRNA levels of ACVR2B in whole blood were quantified using qRT-PCR. Outcome was assessed at 3 years of age using standardised developmental assessment. In total, 171 infants were enrolled: 88 healthy controls, 56 PA and 27 HIE. A statistically significant elevation of median (IQR) ACVR2B was detected in infants with severe HIE compared to moderate/mild HIE, PA and control groups (3.3 (2.94-3.67) vs. 0.91 (0.55-1.21) vs. 0.88 (0.57-1.38) vs. 0.84 (0.74-1.24), p values = 0.04, 0.027 and 0.025, respectively). Although serum activin-A levels were elevated in infants with severe HIE, this elevation did not reach significance. ACVR2B may be a potential novel marker of HIE severity. This is the first study to examine the relationship between activin-A, its receptor AVCR2B and potentially upstream miRNA miR-374a in a cohort of carefully categorised and phenotyped infants. We have shown that miRNA analysis, combined with downstream target exploration, may yield novel biomarkers for the prediction of HIE severity.

Evidence of selection for clones having genetic inactivation of the activin A type II receptor (ACVR2) gene in gastrointestinal cancers.

The activin signaling pathway parallels the transforming growth factor (TGF)-beta pathway. Both use extracellular ligands and cell surface receptors that are structurally and functionally related, as well as the same intracellular mediators (SMADs 2-4) to transmit these signals. Members of both pathways have been characterized previously as tumor suppressor genes on the demonstration of inactivating mutations in human neoplasms, e.g., genetic inactivation of the activin type I receptor was reported recently in pancreatic cancer. Here, we present evidence of selection for mutations of the activin A type II receptor (ACVR2) gene during human gastrointestinal carcinogenesis. Two 8-bp polyadenine tracts of the ACVR2 gene are targets for inactivating frameshift mutations in gastrointestinal neoplasms having microsatellite instability (MSI). These mutations are similar to those of the 10-bp polyadenine tract within the TGF-beta type II receptor (TGFBR2), a well-characterized target of frameshift mutations in the same neoplasms. We identified biallelic mutations of ACVR2 in 25 of 28 MSI colorectal and pancreatic cancers. In addition, a mutation in the ACVR2 gene combined with loss of the wild-type allele was found in a non-MSI pancreatic cancer. This evidence is compatible with a high degree of selection for inactivation of the ACVR2 gene in tumorigenesis, supporting ACVR2 as a candidate tumor suppressor gene in gastrointestinal cancers.

[Gene regulatory mechanism of hairy and enhancer of split related-1 for angiogenesis factors].

OBJECTIVE: To study the roles of hairy and enhancer of split related-1 (HESR-1) in maintenance of the mature, quiescent vessel and angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured. The full-length coding sequence of HESR-1 was cloned into PcDNA3.1 + using standard protocols. HESR-1 specific siRNA was synthesized and cloned into the RNAi-Ready pSIREN-RetroQ ZsGreen Vector. The constructed PcDNA3.1 + HESR-1 plasmid were transfected into HUVEC for the overexpression of HESR-1, and HESR-1-RNAi plasmid were transfected into HUVEC to silence the HESR-1 gene, the expression of KDR, ALK-1 and Ang-1 in HUVEC were analyzed by RT-PCR and Western blot. RESULTS: The expression of KDR was down-regulated and ALK-1 and Ang-1 were up-regulated in HUVEC with the overexpression of HESR-1; The expression of KDR was up-regulated and ALK-1 and Ang-1 were down-regulated the HESR-1 in HUVEC by RNAi. CONCLUSION: HESR-1 may play an important role in maintenance of vessel in quiescent and control angiogenesis by the regulation of the expression of KDR, ALK-1 and Ang-1.

Elevated levels of activin A in heart failure: potential role in myocardial remodeling.

BACKGROUND: Although modulation of inflammatory processes has been suggested as a new treatment modality in heart failure (HF), our knowledge about abnormalities in the cytokine network during HF is still limited. On the basis of a previous cDNA array study examining peripheral blood mononuclear cells from HF patients, we hypothesized a role for activin A, a member of the transforming growth factor (TGF)-beta superfamily, in the pathogenesis of HF. METHODS AND RESULTS: This study had 4 main and novel findings. First, serum levels of activin A were significantly elevated in patients with HF (n=86) compared with healthy control subjects (n=20), with increasing levels according to disease severity as assessed by clinical, hemodynamic, and neurohormonal parameters. Second, compared with control subjects, HF patients, as determined by real-time quantitative reverse transcriptase polymer chain reaction, also had markedly increased gene expression of the activin A subunit activin betaA in T cells but not in monocytes. Third, in a rat model of HF, we demonstrated a concerted induction of the gene expression of activin betaA and activin receptors IA, IB, IIA, and IIB after myocardial infarction. Immunohistochemical analysis localized activin A solely to cardiomyocytes. Finally, activin A markedly increased gene expression of mediators involved in infarction healing and myocardial remodeling (ie, atrial natriuretic peptide, brain natriuretic peptide, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta1, and monocyte chemoattractant protein-1) in neonatal rat cardiomyocytes. CONCLUSIONS: Together with our demonstration of activin A-induced gene expression in neonatal cardiomyocytes of mediators related to myocardial remodeling, the expression pattern of activin A during clinical and experimental HF suggests an involvement of this cytokine in the pathogenesis of HF.

microRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells.

microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

Effect of adenovirus-mediated overexpression of follistatin and extracellular domain of activin receptor type II on gonadotropin secretion in vitro and in vivo.

Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.

Regulation of endometrial adenocarcinoma cell proliferation by Activin-A and its modulation by 17beta-estradiol.

A role for activins in regulating cellular transformation is suggested by the alpha-inhibin knockout mouse in which development of gonadal tumors is associated with elevated activin levels. It was the purpose of the current study to determine whether activin had similar actions on endometrial cell lines, specifically on a well differentiated estrogen-responsive endometrial adenocarcinoma cell line (ISH) and estrogen-unresponsive cells (HEC-50) obtained from a poorly differentiated endometrial adenocarcinoma. Activin was secreted by both adenocarcinoma cell lines. Using reverse transcription-PCR, messenger RNA type I and type II activin receptor subtypes were detected in both cell lines: expression of IB and IIB was approximately three- to fourfold greater in ISH cells than in HEC-50 cells, while activin receptor IA and IIA messenger RNA levels were approximately equal in both cell lines. Activin treatment (30-300 ng/ml) caused a dose- and time-dependent inhibition of ISH cells proliferation and resulted in a significant decrease in Bcl-2 protein and mRNA levels. No difference was observed in Bax expression. There was no significant effect of activin when the cultures of ISH cells were exposed to 17beta-estradiol. In contrast, activin showed a weak, but significant, mitogenic effect on HEC-50 cells without modifications in Bax and Bcl-2 mRNA and protein levels. The results demonstrate that activin is a regulator of endometrial cancer cell growth. 17beta-Estradiol may promote resistance of estrogen-responsive endometrial cancer cells to the growth-retarding effects of activin and one of the mechanisms might be a down-regulation of the activin receptors.

Goat activin receptor type IIB knockdown by artificial microRNAs in vitro.

Activin receptor type IIB (ACVR2B) has been known to negatively regulate the muscle growth through mediating the action of transforming growth factor beta superfamily ligands. Recently, the artificial microRNAs (amiRNAs) which are processed by endogenous miRNA processing machinery have been proposed as promising approach for efficient gene knockdown. We evaluated amiRNAs targeting goat ACVR2B in HEK293T and goat myoblasts cells. The amiRNAs were designed based on the miR-155 backbone and cloned in 5'- and 3'-UTR of GFP reporter gene under the CMV promoter. Although both 5'- and 3'-UTR-amiRNAs vectors showed efficient synthesis of GFP transcripts, amiRNAs in 5'-UTR drastically affected GFP protein synthesis in transfected goat myoblast cells. Among the four amiRNAs targeting ACVR2B derived from either 5'- or 3'-UTR, ami318 showed highest silencing efficiency against exogenously co-expressed ACVR2B in both 293T and goat myoblast cells whereas ami204 showed highest silencing efficiency against endogenous ACVR2B in goat myoblasts cells. The 3'-UTR-derived amiRNA exerted higher knockdown efficiency against endogenous ACVR2B at transcript level whereas 5'-UTR-derived amiRNAs exerted higher knockdown efficiency at protein level. The expression of ACVR2B showed positive correlation with the expression of MYOD (r = 0.744; p = 0.009) and MYOG (r = 0.959; p = 0.000) in the amiRNA-transfected myoblasts. Although both 5'- and 3'-UTR-amiRNA vectors led to substantial induction of interferon response, the magnitude of the response was found to be higher with the 3'-UTR-amiRNA vectors.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

Expression of the TGF-ß-ALK-1 pathway in dura and the outer membrane of chronic subdural hematomas.

Neovascularization of the outer membrane plays a critical role in the development and enlargement of chronic subdural hematomas (CSHs) and vascular endothelial growth factor (VEGF) may promote their progression. However, the precise mechanisms remain to be determined. We focused on the signaling pathway upstream of VEGF, transforming growth factor ß (TGF-ß), and activin receptor-like kinase 1 (ALK-1) to identify the mechanisms underlying the neovascularization of the outer membrane of CSH. Retrospective comparative study was conducted on 15 consecutive patients diagnosed as CSH with burr-hole drainage. Dura and the outer membrane were collected. We immunohistochemically examined the expression of VEGF, integrin-α, TGF-ß, and ALK-1 on the outer membrane and dura of CSH and compared our findings with control samples and the signal intensity of hematomas on computed tomography (CT) scans. VEGF and integrin-α expression was markedly up-regulated in both the dura and outer membrane of CSH, the expression of TGF-ß and ALK-1 in the dura was slightly increased in the dura and markedly up-regulated in the outer membrane. There was no significant correlation between their expression and CT density. Here we first report the expression of TGF-ß and ALK-1 in the outer membrane and dura mater of CSH. We suggest that the TGF-ß-ALK-1 pathway and VEGF affect neovascularization and the progression of CSH.

Papillary thyroid carcinoma with bone formation.

Bone formation is a rarely encountered finding during histological examination of papillary thyroid carcinoma (PTC). This study aimed to analyze clinicopathological parameters in patients with PTC showing bone formation, to document histological features of bone formation in PTC, and to investigate osteogenic proteins. Bone morphogenic protein (BMP)-9 is known as the most potent osteoinductive protein of the BMP subtypes. Recent research suggests that the activin receptor-like kinase (ALK) 1 is an essential cellular receptor that mediates BMP-9-induced osteogenic signaling. A retrospective review of tumor sections from 567 patients with a diagnosis of PTC was performed. Using immunohistochemistry and quantitative real-time polymerase chain reaction, we investigated the expression of ALK1 and BMP-9 in normal thyroid tissue and PTC samples with and without bone formation. Bone formation was found in 13% of patients with PTC. A significant association was seen between bone formation and old age. BMP-9 expression in tumors was increased compared to that in normal thyroid tissues. BMP-9 expression in tumors with bone formation was not significantly different from that in tumors without bone formation. ALK1 expression in tumors with bone formation was increased compared to that in normal thyroid tissue and tumors without bone formation. Our study suggests that upregulation of ALK1 might be an underlying molecular mechanism that explains osteogenesis in PTC.

Constitutively active ALK2 receptor mutants require type II receptor cooperation.

Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease.