SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells.

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells with Elevated Osteogenic Potency by Defined Transcription Factors.

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here, we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9+/RUNX2+ cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium, mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro, but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether, we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model.

HOX genes are frequently dysregulated in human leukaemia with the gene rearrangement between mixed lineage leukaemia (MLL) and partner genes. The resultant MLL fusion proteins are known to mediate leukaemia through disruption of the normal epigenetic regulation at the target gene loci. To elucidate the pathogenic role of MLL fusion proteins in HOX dysregulation in leukaemia, we generated a novel haematopoietic lineage-specific Mll-Een knock-in mouse model using a Cre-mediated inversion strategy. The Mll(Een) (/+) invertor mice developed acute myeloid leukaemia, with organomegaly of the spleen, liver and mesenteric lymph nodes caused by infiltration of blast cells. Using Mll-Een-expressing leukaemic cell lines derived from bone marrow of Mll(Een) (/+) mutant mice, we showed that induction of Hox genes in leukaemic cells was associated with hypomethylated promoter regions and an aberrant active chromatin state at the Hox loci. Knock-down of Prmt1 was insufficient to reverse the active chromatin status and the hypomethylated Hox loci, suggesting that Prmt1-mediated histone arginine methylation was only partially involved in the maintenance of Hox expression in leukaemic cells. Furthermore, in vivo analysis of bone marrow cells of Mll(Een) (/+) mice revealed a Hox expression profile similar to that of wild-type haematopoietic stem cells. The leukaemic Hox profile was highly correlated with aberrant hypomethylation of Hox promoters in the mutant mice, which highlights the importance of DNA methylation in leukaemogenic mechanisms induced by MLL fusion proteins. Our results point to the involvement of dynamic epigenetic regulations in the maintenance of the stem cell-like HOX code that initiates leukaemic stem cells in MLL-rearranged leukaemia. This provides insights for the development of alternative strategies for leukaemia treatment.

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Cyp26b1 mediates differential neurogenicity in axial-specific populations of adult spinal cord progenitor cells.

Utilization of endogenous adult spinal cord progenitor cells (SCPCs) for neuronal regeneration is a promising strategy for spinal cord repair. To mobilize endogenous SCPCs for injury repair, it is necessary to understand their intrinsic properties and to identify signaling factors that can stimulate their neurogenic potential. In this study, we demonstrate that adult mouse SCPCs express distinct combinatorial Hox genes and exhibit axial-specific stem cell properties. Lumbar-derived neurospheres displayed higher primary sphere formation and greater neurogenicity compared with cervical- and thoracic-derived neurospheres. To further understand the mechanisms governing neuronal differentiation of SCPCs from specific axial regions, we examined the neurogenic responses of adult SCPCs to retinoic acid (RA), an essential factor for adult neurogenesis. Although RA is a potent inducer of neuronal differentiation, we found that RA enhanced the generation of neurons specifically in cervical- but not lumbar-derived cells. We further demonstrate that the differential RA response was mediated by the RA-degrading enzyme cytochrome P450 oxidase b1 Cyp26b1. Lumbar cells express high levels of Cyp26b1 and low levels of the RA-synthesizing enzyme retinaldehyde dehydrogenase Raldh2, resulting in limited activation of the RA signaling pathway in these cells. In contrast, low Cyp26b1 expression in cervical spinal cord progenitor cells allows RA signaling to be readily activated upon RA treatment. The intrinsic heterogeneity and signaling factor regulation among adult SCPCs suggest that different niche factor regimens are required for site-specific mobilization of endogenous SCPCs from distinct spatial regions of the spinal cord for injury repair.

Influence of nicotine on the biological activity of rabbit osteoblasts.

OBJECTIVES: To assess the influence of nicotine on the proliferation and gene expression of osteogenic and angiogenic mediators of osteoblasts. MATERIAL AND METHODS: Rabbit primary osteoblasts were exposed to various concentrations of nicotine (0.001, 0.1 and 10 µmol/l). The cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The gene expression of transforming growth factor (TGF)-ß(1), bone morphogenetic protein (BMP)-2, platelet-derived growth factor (PDGF)-AA and vascular endothelial growth factor (VEGF) was evaluated using real-time reverse transcription - polymerase chain reaction. RESULTS: The osteoblast proliferation was inhibited by nicotine at the concentration of 0.001-10 µM at 48 and 72 h of culture, but with no significant effect at 24 h. The expression of TGF-ß(1), BMP-2, PDGF-AA and VEGF was inhibited by nicotine at the concentrations of 0.1 and 10 µM, but with no significant difference at the low concentration of 0.001 µM. CONCLUSIONS: Nicotine suppresses osteoblast proliferation and inhibits the expression of some key osteogenic and angiogenic mediators in the in vitro experimental model. These inhibitory effects of nicotine on the osteoblast activity may reflect, to a certain degree, the overall detrimental effects of tobacco use on the survival rate of dental implants.

Effect of platelet-rich plasma on a rabbit model of nicotine-compromised bone healing.

PURPOSE: This study aimed to evaluate the effects of platelet-rich plasma (PRP) on nicotine-compromised bone healing. MATERIALS AND METHODS: Fifteen adult New Zealand white rabbits were implanted with 1.5-g time-release nicotine pellets. Bilateral mandibular distraction osteogenesis was then performed. Autologous PRP was injected into 1 side of the distraction regenerate, whereas physiologic saline solution was injected into the contralateral side as a control. Five rabbits were killed on day 5 of active distraction, on day 11 of active distraction, and in week 2 of consolidation, respectively. RESULTS: In the PRP the platelet enrichment was 14.63 ± 3.081-fold of that in whole blood. Plain radiography and micro-computed tomography assessment showed no significant difference between the PRP injection and control sides. Histologic examination showed more disorganized distraction tissue on the PRP injection side. CONCLUSIONS: PRP injection at an early stage of active distraction does not significantly enhance bone healing in the nicotine-compromised rabbit model of mandibular lengthening.

Influence of low-dose nicotine on bone healing.

BACKGROUND: Nicotine at a low concentration was suggested as a new topical drug for clinical application. It has been reported to be capable of enhancing skin wound healing. This study was designed to assess the effect of nicotine administration at a low dose on bone regeneration using a rabbit model of mandibular distraction osteogenesis. METHODS: Twenty New Zealand white rabbits were randomly assigned to nicotine group and control group. A total of 0.75 g, 60-day time release, nicotine pellets or placebos were implanted in the neck subcutaneous tissue of the rabbits. The nicotine or placebo exposure time for all the animals was 7 weeks. Unilateral mandibular distraction osteogenesis was performed. Five animals in each group were killed on week 2 and week 4 of consolidation, respectively. The mandibular samples were subjected to radiographic, histologic, and immunohistochemical analysis. RESULTS: Nicotine at low dose showed no significant effect on the expression of bone morphogenetic protein-2 and on the radiodensity of bone regeneration. However, the delayed bone healing was detected in the nicotine group by histologic examination. CONCLUSIONS: These findings imply a potential risk of compromised bone healing in patients taking nicotine medication. Further clinical studies are necessary to assess the risk of nicotine medication on reconstructive surgery.

Effect of nicotine on gene expression of angiogenic and osteogenic factors in a rabbit model of bone regeneration.

PURPOSE: This study aims to evaluate the influence of nicotine on the gene expression of osteogenic and angiogenic factors in bone regeneration by use of a nicotine-compromised rabbit model of mandibular lengthening. MATERIALS AND METHODS: Thirty adult New Zealand white rabbits were randomly assigned to the nicotine group or the control group. The total nicotine or placebo exposure time for all animals was 7 weeks. Unilateral mandibular distraction osteogenesis was performed. Five animals in each group were sacrificed at day 5, day 11, and day 18, respectively, after commencement of active distraction. The distraction regenerate samples were harvested, and the messenger ribonucleic acid expression of bone transforming growth factor beta(1), platelet-derived growth factor A, and basic fibroblast growth factor was assayed by real-time polymerase chain reaction analysis. RESULTS: The messenger ribonucleic acid expression of transforming growth factor beta(1), platelet-derived growth factor A, and basic fibroblast growth factor was significantly inhibited by nicotine exposure at a variety of time points. CONCLUSIONS: The presence of nicotine inhibited the gene expression of angiogenic and osteogenic factors resulting in compromised bone regeneration.

Uncoupled angiogenesis and osteogenesis in nicotine-compromised bone healing.

Nicotine is the main chemical component responsible for tobacco addiction. This study aimed to evaluate the influence of nicotine on angiogenesis and osteogenesis and the associated expression of angiogenic and osteogenic mediators during bone healing. Forty-eight adult New Zealand White rabbits were randomly assigned to a nicotine group and a control group. Nicotine pellets (1.5 g, 60-day time release) or placebo pellets were implanted in the neck subcutaneous tissue. The nicotine or placebo exposure time for all the animals was 7 weeks. Unilateral mandibular distraction osteogenesis was performed. Eight animals in each group were euthanized on day 5, day 11 of active distraction, and week 1 of consolidation, respectively. The mandibular samples were subjected to radiographic, histologic, immunohistochemical, and real-time reverse-transcriptase polymerase chain reaction examinations. Nicotine exposure upregulated the expression of hypoxia inducible factor 1alpha and vascular endothelial growth factor and enhanced angiogenesis but inhibited the expression of bone morphogenetic protein 2 and impaired bone healing. The results indicate that nicotine decouples angiogenesis and osteogenesis in this rabbit model of distraction osteogenesis, and the enhanced angiogenesis cannot compensate for the adverse effects of nicotine on bone healing.

Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional collagen constructs.

Mechanical signal is important for regulating cellular activities, including proliferation, metabolism, matrix production, and orientation. Bioreactors with loading functions can be used to precondition cells in three-dimensional (3D) constructs so as to study the cellular responses to mechanical stimulation. However, full-scale bioreactor is not always an affordable option considering the high cost of equipment and the liter-sized medium with serum and growth factor supplements. In this study, a custom-built loading system was developed by coupling a conventional camera-equipped inverted research microscope with two micromanipulators. The system was programmed to deliver either cyclic compressive loading with different frequencies or static compressive loading for 1 week to investigate the cellular responses of human mesenchymal stem cells (hMSCs) entrapped in a 3D construct consists of reconstituted collagen fibers. Cellular properties, including their alignment, cytoskeleton, and cell metabolism, and properties of matrix molecules, such as collagen fiber alignment and glycosaminoglycan deposition, were evaluated. Using a MatLab-based image analysis program, reorientation of the entrapped cells from a random distribution to a preferred alignment along the loading direction in constructs with both static and cyclic compression has been demonstrated, but no such alignment was found in the free-floating controls. Fluorescent staining on filamentous actin cytoskeleton also confirmed the finding. Nevertheless, the collagen fiber meshwork entrapping the hMSCs remained randomly distributed, and no change in cellular metabolism and glycosaminoglycans production was noted. The current study provides a simple and affordable option toward setting up a mechanoregulation facility based on existing laboratory equipment and sheds new insights on the effect of mechanical loading on the alignment of hMSCs in 3D collagen constructs.

Suppression of lung tumor growth and metastasis in mice by adeno-associated virus-mediated expression of vasostatin.

PURPOSE: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness of vasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS) as a gene therapy approach for lung cancer treatment. EXPERIMENTAL DESIGN: We used rAAV2/5 to deliver vasostatin intratumorally or systemically in different mouse lung tumor models--subcutaneous, orthotopic xenograft, and spontaneous metastasis lung tumor models. The therapeutic efficacy of rAAV2/5-VAS was determined by monitoring tumor volume, survival rate, and degree of neovascularization after treatment in these models. RESULTS: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinoma cells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonary metastases. The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumor A549 xenograft correlated with a reduced number of capillary vessels in tumors. In the orthotopic xenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymph nodes and contralateral lung. Furthermore, treatment of immunocompetent mice in the spontaneous lung metastases model with rAAV2/5-VAS after primary tumor excision prolonged their median survival from 21 to 51.5 days. CONCLUSION: Our results show the effectiveness of rAAV2/5-VAS as an angiogenesis inhibitor in suppressing tumor growth during different stages of tumor progression, validating the application of rAAV2/5-VAS gene therapy in treatment against lung cancer.

Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells.

Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.

Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow.

OBJECTIVE: Hox genes are involved in hematopoietic lineage commitment and differentiation. In this study, we investigated the roles of Hoxb3 in hematopoiesis by examining the phenotypes of a Hoxb3 knockout mutant mouse line. RESULTS: Despite previous reports describing the apparently normal phenotype of these mutant mice, we found that by 6 months of age, Hoxb3(-/-) mice began to exhibit significantly impaired B lymphopoiesis in the bone marrow (BM). The cellularity was reduced by 30% in mutant BM compared to age- and sex-matched heterozygous and wild-type controls. The population size of B220(+)CD43(+) progenitor B cells showed a twofold reduction while that of B220(+)CD43(-)IgM(-) precursor B cells was decreased fivefold. Sorting-purified Hoxb3(-/-) progenitor B cells displayed significantly reduced proliferative response to IL-7 in culture, consistent with our findings of reduced IL-7 receptor expression in Hoxb3(-/-) progenitor B cells. However, the peripheral B cell pool in the spleen of Hoxb3(-/-) mice was maintained with a similar size as in wild-type littermates. CONCLUSION: Analysis of T-cell development in the thymus and B1 cell compartment in the peritoneal cavity showed no significant changes. Thus, our findings suggest that the Hoxb3 gene plays an essential role in regulating B lymphopoiesis in the BM of adult mice.

Overexpression of soluble TRAIL induces apoptosis in human lung adenocarcinoma and inhibits growth of tumor xenografts in nude mice.

Recombinant adeno-associated virus 2/5 (rAAV2/5), a hybrid rAAV-2 with AAV-5 capsid, seems to be a very efficient delivery vector for the transduction of the lung adenocarcinoma cell line A549. Infection of the A549 cell line with a rAAV2/5 vector encoding the extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, amino acids 114-281) resulted in secretion of soluble TRAIL (sTRAIL) and induction of apoptosis in these cells. rAAV2/5-sTRAIL mediated delivery and stable expression of sTRAIL resulted in the presence of the trimeric form of sTRAIL in sera of nude mice that were implanted with s.c. or orthotopic A549 tumors. The rAAV2/5-sTRAIL transduction of the tumors resulted in a statistically significant reduction in tumor growth and prolonged survival of the tumor-bearing animals. Primary cell culture, histologic examination of the tumors, and serum analyses showed the absence of detectable TRAIL-induced toxicity in normal tissues including the liver. The successful inhibition of lung cancer growth and the absence of detectable toxicity suggest a putative role for rAAV2/5-sTRAIL(114-281) in the therapy of lung cancer.

TTF-1 and RET promoter SNPs: regulation of RET transcription in Hirschsprung's disease.

Single nucleotide polymorphisms (SNPs) of the coding regions of receptor tyrosine kinase gene (RET) are associated with Hirschsprung's disease (HSCR, aganglionic megacolon). These SNPs, individually or combined, may act as a low penetrance susceptibility locus and/or be in linkage disequilibrium (LD) with another susceptibility locus located in RET regulatory regions. Because two RET promoter SNPs have been found associated with HSCR, in LD with HSCR-associated RET coding region haplotypes, their implication in the transcriptional regulation of RET is of major interest. Analysis of 172 sporadic HSCR patients also revealed the presence of HSCR-associated RET promoter SNPs in LD with the main coding region RET haplotype observed in Chinese patients. By using a weighted logistic regression approach, we determined that of all SNPs tested in our study, the promoter SNPs are the most correlated to the disease. Functional analysis of the RET promoter SNPs in the context of additional 5' regulatory regions demonstrated that the HSCR-associated alleles decrease RET transcription. These SNPs overlap a TTF-1 binding site and TTF-1-activated RET transcription is also decreased by the HSCR-associated SNPs. Moreover, we identified an HSCR patient with a Gly322Ser TTF-1 mutation that compromises activation of transcription from HSCR-associated RET promoter haplotypes. Interestingly, we show that the pattern of RET and TTF-1 expression is coincident in developing human gut. We also present a detailed profile of the RET gene in our population, which provides an insight into the higher incidence of the disease in China.