Enam expression is regulated by Msx2.

BACKGROUND: The precise formation of mineralized dental tissues such as enamel and/or dentin require tight transcriptional control of the secretion of matrix proteins. Here, we have investigated the transcriptional regulation of the second most prominent enamel matrix protein, enamelin, and its regulation through the major odontogenic transcription factor, MSX2. RESULTS: Using in vitro and in vivo approaches, we identified that (a) Enam expression is reduced in the Msx2 mouse mutant pre-secretory and secretory ameloblasts, (b) Enam is an early response gene whose expression is under the control of Msx2, (c) Msx2 binds to Enam promoter in vitro, suggesting that enam is a direct target for Msx2 and that (d) Msx2 alone represses Enam gene expression. CONCLUSIONS: Collectively, these results illustrate that Enam gene expression is controlled by Msx2 in a spatio-temporal manner. They also suggest that Msx2 may interact with other transcription factors to control spatial and temporal expression of Enam and hence amelogenesis and enamel biomineralization.

The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation.

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.

The prolyl isomerase Pin1 increases ß-cell proliferation and enhances insulin secretion.

The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic ß cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic ß cells, we generated ß-cell-specific Pin1 KO (ßPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the ßPin1 KO mice. Pin1 enhanced pancreatic ß-cell proliferation, as indicated by a reduced ß-cell mass in ßPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic ß-cell growth in the ßPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO ß cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca2+ concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca2+ influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca2+ influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic ß cells and that Pin1 both promotes ß-cell proliferation and activates insulin secretion.

Mitochondrial Activity and Unfolded Protein Response are Required for Neutrophil Differentiation.

BACKGROUND/AIMS: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in hematopoietic differentiation. However, the mechanistic linkage between ER stress/UPR and hematopoietic differentiation remains unclear. METHODS: We used bipotent HL-60 cells as an in vitro hematopoietic differentiation system to investigate the role of ER stress and UPR activity in neutrophil and macrophage differentiation. RESULTS: The in vitro differentiation analysis revealed that ER stress decreased during both neutrophil and macrophage differentiations, and the activities of PERK and ATF6 were decreased and that of IRE1α was increased during neutrophil differentiation in a stage-specific manner. By contrast, the activities of ATF6 and ATF4 decreased during macrophage differentiation. When the cells were treated with oligomycin, the expression of CD11b, a myelocytic differentiation marker, and morphological differentiation were suppressed, and XBP-1 activation was inhibited during neutrophil differentiation, whereas CD11b expression was maintained, and morphological differentiation was not obviously affected during macrophage differentiation. CONCLUSION: In this study, we demonstrated that neutrophil differentiation is regulated by ER stress/UPR that is supported by mitochondrial ATP supply, in which IRE1α-XBP1 activation is essential. Our findings provide the evidence that mitochondrial energy metabolism may play a critical role in neutrophil differentiation.

Porphyromonas gingivalis infection alters microRNA composition in extracellular vesicles.

OBJECTIVES: Periodontitis, commonly associated with Porphyromonas gingivalis (Pg), involves intricate alterations of oral intercellular interactions, in which extracellular vesicles (EVs) play a pivotal role. The understanding of the miRNA profiles in the EVs derived from Pg-infected cells (Pg-EVs) remains incomplete despite acknowledging their importance in intercellular communication during periodontitis. Therefore, our objective was to identify and characterize the miRNAs enriched in Pg-EVs. METHODS: Microarray analysis was conducted to examine the miRNA profiles in the EVs derived from Pg-infected THP-1 cells. We compared the identified miRNAs with those upregulated in the EVs after stimulation with LPS. Additionally, we explored how inhibiting TLR signaling during Pg infection affects the transcription of specific miRNAs. We investigated the unique sequence motifs specific to the miRNAs concentrated in Pg-EVs. RESULTS: The levels of eleven miRNAs, including miR-155, were increased in Pg-EVs compared with those elevated after LPS stimulation. The Pg-induced miR-155 upregulation via TLR2 but not TLR4 signaling suggests the influence of TLR signaling on the miRNA composition of EVs. Furthermore, the miRNAs upregulated in Pg-EVs contained AGAGGG and GRGGSGC sequence motifs. CONCLUSIONS: Our findings demonstrate that Pg-induced alterations in EV-containing miRNA composition occur in a TLR4-independent manner. Notably, the concentrated miRNAs in Pg-EVs harbor specific motifs with a high G + C content within their sequences. The upregulation of specific miRNAs in EVs under infectious conditions suggests the influence of both innate immune receptor signals and miRNA sequence characteristics.

Involvement of TGFBI-TAGLN axis in cancer stem cell property of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.

Involvement of the OTUB1-YAP1 axis in driving malignant behaviors of head and neck squamous cell carcinoma.

BACKGROUND: Comprehending the molecular mechanisms underlying head and neck squamous cell carcinoma (HNSCC) is vital for the development of effective treatment strategies. Deubiquitinating enzymes (DUBs), which regulate ubiquitin-dependent pathways, are potential targets for cancer therapy because of their structural advantages. Here we aimed to identify a potential target for HNSCC treatment among DUBs. METHODS: A screening process was conducted using RNA sequencing data and clinical information from HNSCC patients in the TCGA database. A panel of 88 DUBs was analyzed to identify those associated with poor prognosis. Subsequently, HNSCC cells were modified to overexpress specific DUBs, and their effects on cell proliferation and invasion were evaluated. In vivo experiments were performed to validate the findings. RESULTS: In HNSCC patients, USP10, USP14, OTUB1, and STAMBP among the screened DUBs were associated with a poor prognosis. Among them, OTUB1 showed the most aggressive characteristics in both in vitro and in vivo experiments. Additionally, OTUB1 regulated the stability and nuclear localization of YAP1, a substrate involved in cell proliferation and invasion. Notably, OTUB1 expression exhibited a positive correlation with the HNSCC-YAP score in HNSCC cells. CONCLUSIONS: This study highlights the critical role of OTUB1 in HNSCC progression via modulating YAP1. Targeting the OTUB1-YAP1 axis holds promise as a potential therapeutic strategy for HNSCC treatment.

miR-155-5p can be involved in acquisition of osseointegration on titanium surface.

Dental implants made of titanium are commonly used. Although titanium implants succeed by osseointegration with bone, the detailed molecular mechanism of osseointegration is unclear. To clarify the involvement of microRNA (miRNA) in the acquisition of osseointegration on titanium, here we compared the miRNA expression profiles of mouse osteoblast-like cells (MC3T3-E1) cultured on titanium-, gold-, and stainless steel-coating glass dishes by microarray analysis. Three kinds of metals, namely titanium, gold, and stainless steel, were coated on the surface of the glass dishes by sputtering with similar roughness and shape of their surface. After MC3T3-E1 cells were cultured on the dishes without coating or coating with titanium, gold, or stainless steel for 6 h, total RNA was extracted, and miRNA expression was analyzed by microarray. To confirm the expression of the selected miRNA during osteogenic differentiation of MC3T3-E1 cells, real-time PCR analysis was performed. Furthermore, the effects of selected miRNA were examined by ectopic overexpression in MC3T3-E1 cells. The microarray analysis revealed that the expressions of miR-155-5p and miR-7023-3p were significantly increased in MC3T3-E1 cells cultured on titanium-coating glass dishes, compared to non-coating, gold-, and stainless steel-coating glass dishes. Interestingly, miR-155-5p was upregulated during osteogenic differentiation of MC3T3-E1 cells. Furthermore, overexpression of miR-155-5p enhanced the expression of Runx2 and Col1a1. In this study, miR-155-5p may be involved in the acquisition of osseointegration on titanium implant via upregulating osteogenic differentiation-related genes.

Redefining GBA gene structure unveils the ability of Cap-independent, IRES-dependent gene regulation.

Glucosylceramide is the primary molecule of glycosphingolipids, and its metabolic regulation is crucial for life. Defects in the catabolizing enzyme, glucocerebrosidase (GCase), cause a lysosomal storage disorder known as Gaucher disease. However, the genetic regulation of GCase has not been fully understood. Here we show the redefined structure of the GCase coding gene (GBA), and clarify the regulatory mechanisms of its transcription and translation. First, alternative uses of the two GBA gene promoters were identified in fibroblasts and HL60-derived macrophages. Intriguingly, both GBA transcripts and GCase activities were induced in macrophages but not in neutrophils. Second, we observed cap-independent translation occurs via unique internal ribosome entry site activities in first promoter-driven GBA transcripts. Third, the reciprocal expression was observed in GBA and miR22-3p versus GBAP1 transcripts before and after HL60-induced macrophage differentiation. Nevertheless, these findings clearly demonstrate novel cell-type-specific GBA gene expression regulatory mechanisms, providing new insights into GCase biology.

The role of partial-EMT in the progression of head and neck squamous cell carcinoma.

BACKGROUND: It is well-established that cervical lymph node metastasis is the most important prognostic factor in head and neck squamous cell carcinoma (HNSCC). Cancer cells invade the underlying stroma during metastasis by breaching the basement membrane. HIGHLIGHT: The ability to metastasize is a key hallmark of cancer progression and this characteristic can be attained by undergoing epithelial-to-mesenchymal transition (EMT). EMT is a biological process in which epithelial cells lose their epithelial features and gain mesenchymal features. Recent evidence reports the intermediate state in the induction of EMT and partial-EMT. Notably, the partial-EMT phenotype is more aggressive than the complete EMT phenotype. However, the role of partial-EMT is not fully understood. CONCLUSION: In this review, we highlight the features of partial-EMT in HNSCC by summarizing previous studies. Moreover, we discuss the therapeutic potential for targeting partial-EMT.

Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis.

Distinguishing oncogenic mutations from variants of unknown significance (VUS) is critical for precision cancer medicine. Here, computational modeling of 71,756 RET variants for positive selection together with functional assays of 110 representative variants identified a three-dimensional cluster of VUSs carried by multiple human cancers that cause amino acid substitutions in the calmodulin-like motif (CaLM) of RET. Molecular dynamics simulations indicated that CaLM mutations decrease interactions between Ca2+ and its surrounding residues and induce conformational distortion of the RET cysteine-rich domain containing the CaLM. RET-CaLM mutations caused ligand-independent constitutive activation of RET kinase by homodimerization mediated by illegitimate disulfide bond formation. RET-CaLM mutants possessed oncogenic and tumorigenic activities that could be suppressed by tyrosine kinase inhibitors targeting RET. This study identifies calcium-binding ablating mutations as a novel type of oncogenic mutation of RET and indicates that in silico-driven annotation of VUSs of druggable oncogenes is a promising strategy to identify targetable driver mutations. SIGNIFICANCE: Comprehensive proteogenomic and in silico analyses of a vast number of VUSs identify a novel set of oncogenic and druggable mutations in the well-characterized RET oncogene.

Possible linkage of SP6 transcriptional activity with amelogenesis by protein stabilization.

Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

A New Induction Method for the Controlled Differentiation of Human-Induced Pluripotent Stem Cells Using Frozen Sections.

Considering that every tissue/organ has the most suitable microenvironment for its functional cells, controlling induced pluripotent stem cell (iPSC) differentiation by culture on frozen sections having a suitable microenvironment is possible. Induced PSCs were cultured on frozen sections of the liver, the brain, the spinal cord, and cover glasses (control) for 9 days. The iPSCs cultured on the sections of the liver resembled hepatocytes, whereas those on sections of the brain and the spinal cord resembled neuronal cells. The percentage of hepatocytic marker-positive cells in the iPSCs cultured on the sections of the liver was statistically higher than that of those in the iPSCs cultured on the sections of the brain and the spinal cord or on cover glasses. In contrast, the iPSCs cultured on the sections of the brain and the spinal cord revealed a high percentage of neural marker-positive cells. Thus, iPSCs can be differentiated into a specific cell lineage in response to specific factors within frozen sections of tissues/organs. Differentiation efficacy of the frozen sections markedly differed between the iPSC clones. Therefore, our induction method could be simple and effective for evaluating the iPSC quality.

Gene-expression profile reveals the genetic and acquired phenotypes of hyperactive mutant SPORTS rat.

Spontaneously Running Tokushima Shikoku (SPORTS) rat is a hyperactive rat strain. However, the causative mutation of this phenotype has not yet been identified. To investigate the molecular basis for the unique phenotype of SPORTS rats, we examined gene-expression profiles by microarray analyses. Among adenylate kinase isozymes that maintain the homeostasis of cellular adenine nucleotide composition in the cell, only adenylate kinase 1 is highly up-regulated in both exercised and sedentary SPORTS rats compared with wild-type (WT) rats, 5.5-fold and 3.3-fold, respectively. Further comparative analyses revealed that genes involved in glucose metabolism were up-regulated in skeletal muscle tissue of exercised SPORTS rats compared with sedentary mutants, whereas genes related to extracellular matrix or region were down-regulated compared with WT rats. In brain tissue of sedentary SPORTS rats, genes associated with defense and catecholamine metabolism were highly expressed compared with WT rats. These findings suggest that genetic mutation(s) in SPORTS rat remodels metabolic demands through differentially regulating gene expression regardless of exercise. Therefore, the SPORTS rats are useful animal model not only for further examining the effects of exercise on metabolism but also for deeply studying the molecular basis how mutation affect the psychological motivation with spontaneous voluntary exercise phenotype. J. Med. Invest. 67 : 51-61, February, 2020.

An Msx2-Sp6-Follistatin Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis.

BACKGROUND: Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis. METHODS: Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites. RESULTS: Using the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 kb upstream of the murine Sp6 promoter region. By chIP, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2. CONCLUSION: Collectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

Bronchioalveolar stem cells derived from mouse-induced pluripotent stem cells promote airway epithelium regeneration.

BACKGROUND: Bronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles. METHODS: BASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated. RESULTS: Flow cytometric analysis revealed that BASCs represented ~ 7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs. CONCLUSIONS: Mouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.

Sp6 downregulation of follistatin gene expression in ameloblasts.

Sp6 is a member of the Sp family of transcription factors that regulate a wide range of cellular functions, such as cell growth and differentiation. Sp6, also called epiprofin, is specifically expressed in tooth germ, limb bud, and hair follicle, but there is little information on its function.To investigate the possible role of Sp6 in tooth development, first we established an Sp6-overproducing clone, CHA9, and analyzed the features of the cell, including cell proliferation and gene expression. The parental cells of CHA9 are the ameloblast-lineage G5 cells that we previously established from rat dental epithelia of lower incisor. Sp6 overproduction accelerated cell proliferation and induced the expression of ameloblastin mRNA, a marker of ameloblast differentiation. Second, we performed genome-wide screening of Sp6 target genes by microarray analysis. Out of a total 20,450 genes, 448 genes were up-regulated and 500 genes were down-regulated by Sp6. We found the expression of follistatin, a BMP antagonist, to be 22.4-fold lower in CHA9 than in control cells. Transfection of the Sp6-antisense construct into CHA9 cells restored follistatin expression back to equivalent levels seen in control cells, indicating that Sp6 regulates follistatin gene expression in ameloblasts. Our findings demonstrate that the follistatin gene is one of the Sp6 target genes in ameloblasts and suggest that Sp6 promotes amelogenesis through inhibition of follistatin gene expression.

Deciphering defective amelogenesis using in vitro culture systems.

The conventional two-dimensional (2D) in vitro culture system is frequently used to analyze the gene expression with or without extracellular signals. However, the cells derived from primary culture and cell lines frequently deviate the gene expression profile compared to the corresponding in vivo samples, which sometimes misleads the actual gene regulation in vivo. To overcome this gap, we developed the comparative 2D and 3D in vitro culture systems and applied them to the genetic study of amelogenesis imperfecta (AI) as a model. Recently, we found specificity protein 6 (Sp6) mutation in an autosomal-recessive AI rat that was previously named AMI. We constructed 3D structure of ARE-B30 cells (AMI-derived rat dental epithelial cells) or G5 (control wild type cells) combined with RPC-C2A cells (rat pulp cell line) separated by the collagen membrane, while in 2D structure, ARE-B30 or G5 was cultured with or without the collagen membrane. Comparative analysis of amelogenesis-related gene expression in ARE-B30 and G5 using our 2D and 3D in vitro systems revealed distinct expression profiles, showing the causative outcomes. Bone morphogenetic protein 2 and follistatin were reciprocally expressed in G5, but not in ARE-B30 cells. All-or-none expression of amelotin, kallikrein-related peptidase 4, and nerve growth factor receptor was observed in both cell types. In conclusion, our in vitro culture systems detected the phenotypical differences in the expression of the stage-specific amelogenesis-related genes. Parallel analysis with 2D and 3D culture systems may provide a platform to understand the molecular basis for defective amelogenesis caused by Sp6 mutation.

Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation.

This study explored the functions of the signal transducers and activators of transcription 5a and 5b (referred to as Stat5 here) during different stages of mouse mammary gland development by using conditional gene inactivation. Mammary gland morphogenesis includes cell specification, proliferation and differentiation during pregnancy, cell survival and maintenance of differentiation throughout lactation, and cell death during involution. Stat5 is activated by prolactin, and its presence is mandatory for the proliferation and differentiation of mammary epithelium during pregnancy. To address the question of whether Stat5 is also necessary for the maintenance and survival of the differentiated epithelium, the two genes were deleted at different time points. The 110-kb Stat5 locus in the mouse was bracketed with loxP sites, and its deletion was accomplished by using two Cre-expressing transgenic lines. Loss of Stat5 prior to pregnancy prevented epithelial proliferation and differentiation. Deletion of Stat5 during pregnancy, after mammary epithelium had entered Stat5-mediated differentiation, resulted in premature cell death, indicating that at this stage epithelial cell proliferation, differentiation, and survival require Stat5.

Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium.

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.