Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin-Binding Motif.

The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells' ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of ß-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/ß-catenin signaling.

Deployment of the Ophthalmic and Facial Angiosomes in the Upper Nose Overlaying the Nasal Bones.

BACKGROUND: Nasal filler placement is associated with a high risk of blindness. The arterial supply to the upper nose overlaying the nasal bones is poorly understood. OBJECTIVES: The aim of this study was to visualize and analyze the deployment of the ophthalmic and facial angiosomes in the upper nose to help prevent blindness following nasal filler injections. METHODS: The arterial systems of 62 cadaveric heads were filled with lead oxide contrast agent, and computed tomography (CT) images were acquired and reconstructed in 3 dimensions. RESULTS: Twenty-six of the cadaveric noses examined demonstrated clear CT images of the facial and ophthalmic angiosomes in the upper nose. The Type 1 upper nose (15.4%) is supplied by 2 independent ophthalmic angiosomes that communicate indirectly through a choke anastomosis. The Type 2 upper nose (38.5%) is supplied by 2 ophthalmic angiosomes with a true anastomosis between them. The Type 3 upper nose (46.1%) is supplied by both ophthalmic and facial angiosomes with true anastomoses across the dorsal midline. These true anastomoses are mediated by the radix arcade in 46% of the noses and involve the dorsal nasal artery in 65% of the cases. The anastomoses all cross the upper dorsal midline and are directly linked to the ophthalmic angiosome. CONCLUSIONS: The deployment and anastomosis of the facial and ophthalmic angiosomes in the upper nose fall into 3 major patterns. About 85% of the noses have true anastomotic arteries that cross the upper dorsal midline and are directly linked to the ophthalmic circulation. Dorsum filler injection poses a significant risk of blindness.

Mouse Ptchd3 is a non-essential gene.

Mouse Ptchd3 (patched domain containing 3) was previously identified as a male germ-cell specific gene. The protein product of this gene has been found on the surface of mouse, rat and human sperm. Since Ptchd3 contains a conserved patched domain, we hypothesize that it functions as a membrane receptor for the hedgehog ligand. Herein, we used a Ptchd3 knockout mouse model to study its function in mouse development and spermatogenesis. We found that Ptchd3 knockout mice were born and lived normally. The fertility and sperm production of knockout males were not changed. Moreover, our data indicated that the expression levels of several hedgehog signaling genes were not affected in mutant testis. Taken together, these findings demonstrate that Ptchd3 is a non-essential gene in mouse development and spermatogenesis.

A caveolin binding motif in Na/K-ATPase is required for stem cell differentiation and organogenesis in mammals and C. elegans.

Several signaling events have been recognized as essential for regulating cell lineage specification and organogenesis in animals. We find that the gain of an amino-terminal caveolin binding motif (CBM) in the α subunit of the Na/K-adenosine triphosphatase (ATPase) (NKA) is required for the early stages of organogenesis in both mice and Caenorhabditis elegans. The evolutionary gain of the CBM occurred at the same time as the acquisition of the binding sites for Na+/K+. Loss of this CBM does not affect cell lineage specification or the initiation of organogenesis, but arrests further organ development. Mechanistically, this CBM is essential for the dynamic operation of Wnt and the timely up-regulation of transcriptional factors during organogenesis. These results indicate that the NKA was evolved as a dual functional protein that works in concert with Wnt as a hitherto unrecognized common mechanism to enable stem cell differentiation and organogenesis in multicellular organisms within the animal kingdom.

Mouse Ptchd3 is a non-essential gene.

Mouse Ptchd3 (patched domain containing 3) was previously identified as a male germ-cell specific gene. The protein product of this gene has been found on the surface of mouse, rat and human sperm. Since Ptchd3 contains a conserved patched domain, we hypothesize that it functions as a membrane receptor for the hedgehog ligand. Herein, we used a Ptchd3 knockout mouse model to study its function in mouse development and spermatogenesis. We found that Ptchd3 knockout mice were born and lived normally. The fertility and sperm production of knockout males were not changed. Moreover, our data indicated that the expression levels of several hedgehog signaling genes were not affected in mutant testis. Taken together, these findings demonstrate that Ptchd3 is a non-essential gene in mouse development and spermatogenesis.

High-Throughput Screening of Full-Face Clinically Relevant Arterial Variations Using Three-Dimensional Postmortem Computed Tomography.

BACKGROUND: Vascular complications resulting from intravascular filler injection and embolism are major safety concerns for facial filler injection. It is essential to systematically screen full-face arterial variations and help design evidence-based safe filler injection protocols. METHODS: The carotid arteries of 22 cadaveric heads were infused with adequate lead oxide contrast. The facial and superficial temporal arteries of another 12 cadaveric heads were injected with the contrast in a sequential order. A computed tomographic scan was acquired after each contrast injection, and each three-dimensional computed tomographic scan was reconstructed using validated algorithms. RESULTS: Three-dimensional computed tomography clearly demonstrated the course, relative depth, and anastomosis of all major arteries in 63 qualified hemifaces. The ophthalmic angiosome consistently deploys two distinctive layers of branch arteries to the forehead. The superficial temporal and superior palpebral arteries run along the preauricular and superior palpebral creases, respectively. The study found that 74.6 percent of the hemifaces had nasolabial trunks coursing along the nasolabial crease, and that 50.8 percent of the hemifaces had infraorbital trunks that ran through the infraorbital region. Fifty percent of the angular arteries were the direct anastomotic channels between the facial and ophthalmic angiosomes, and 29.2 percent of the angular arteries were members of the ophthalmic angiosomes. CONCLUSIONS: Full-face arterial variations were mapped using postmortem three-dimensional computed tomography. Facial creases were in general correlated with underlying deep arteries. Facial and angular artery variations were identified at high resolution, and reclassified into clinically relevant types to guide medical practice.

Efficacy of Retrobulbar Hyaluronidase Injection for Vision Loss Resulting from Hyaluronic Acid Filler Embolization.

BACKGROUND: Vision loss is a rare but serious complication of facial hyaluronic acid (HA) filler injection, for which there is no proven rescue therapy. Retrobulbar hyaluronidase injection is advocated by many plastic surgeons as an emergency treatment, but has not been carefully assessed for its efficacy. OBJECTIVES: To evaluate the efficacy of retrobulbar hyaluronidase injection as a rescue treatment for vision loss caused by HA filler embolization. METHODS: Patients with vision loss caused by HA filler embolization were treated with retrobulbar hyaluronidase injection. Their visual acuity and fundoscopic images before and after treatment were analyzed for efficacy assessment. RESULTS: One patient with branch retinal artery occlusion (BRAO), one patient with posterior ischemic optic neuropathy (PION), one patient with ophthalmic artery occlusion, and one patient with both BRAO and PION were treated with one or two retrobulbar injections of 1500 or 3000 units hyaluronidase. No patients demonstrated substantial retinal artery recanalization or vision acuity improvement after treatment. CONCLUSIONS: One or two retrobulbar injections of 1500 to 3000 IU hyaluronidase are unable to recanalize retinal artery occlusion or improve the visual outcome of patients who presented with vision loss caused by HA filler embolization at least four hours after onset. LEVEL OF EVIDENCE: 4.

Clinical Outcomes of Impending Nasal Skin Necrosis Related to Nose and Nasolabial Fold Augmentation with Hyaluronic Acid Fillers.

BACKGROUND: Although there are several case reports of facial skin ischemia/necrosis caused by hyaluronic acid filler injections, no systematic study of the clinical outcomes of a series of cases with this complication has been reported. METHODS: The authors report a study of 20 consecutive patients who developed impending nasal skin necrosis as a primary concern, after nose and/or nasolabial fold augmentation with hyaluronic acid fillers. The authors retrospectively reviewed the clinical outcomes and the risk factors for this complication using case-control analysis. RESULTS: Seven patients (35 percent) developed full skin necrosis, and 13 patients (65 percent) recovered fully after combination treatment with hyaluronidase. Although the two groups had similar age, sex, filler injection sites, and treatment for the complication, 85 percent of the patients in the full skin necrosis group were late presenters who did not receive the combination treatment with hyaluronidase within 2 days after the vascular complication first appeared. In contrast, just 15 percent of the patients in the full recovery group were late presenters (p = 0.004). CONCLUSIONS: Nose and nasolabial fold augmentations with hyaluronic acid fillers can lead to impending nasal skin necrosis, possibly caused by intravascular embolism and/or extravascular compression. The key for preventing the skin ischemia from progressing to necrosis is to identify and treat the ischemia as early as possible. Early (<2 days) combination treatment with hyaluronidase is associated with the full resolution of the complication. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

PITX2 associates with PTIP-containing histone H3 lysine 4 methyltransferase complex.

Pituitary homeobox 2 (PITX2), a Paired-like homeodomain transcription factor and a downstream effector of Wnt/ß-catenin signaling, plays substantial roles in embryonic development and human disorders. The mechanism of its functions, however, is not fully understood. In this study, we demonstrated that PITX2 associated with histone H3 lysine 4 (H3K4) methyltransferase (HKMT) mixed-lineage leukemia 4 (MLL4/KMT2D), Pax transactivation domain-interacting protein (PTIP), and other H3K4·HKMT core subunits. This association of PITX2 with H3K4·HKMT complex was dependent on PITX2's homeodomain. Consistently, the PITX2 protein complex was shown to possess H3K4·HKMT activity. Furthermore, the chromatin immunoprecipitation result revealed co-occupancy of PITX2 and PTIP on the promoter of the PITX2's transcriptional target. Taken together, our data provide new mechanistic perspectives on PITX2's functions and its related biological processes.

Cyclin A1 is a transcriptional target of PITX2 and overexpressed in papillary thyroid carcinoma.

Physiological expression of cyclin A1, a unique cell cycle regulator essential for spermatogenesis, is predominantly restricted in male germ cells. Outstandingly, previous studies have also demonstrated the abnormal expression of cyclin A1 in various human tumors. How male germ cell-specific cyclin A1 is transcriptionally activated in tumor cells, however, is elusive. To begin to understand the molecular mechanisms governing the ectopic expression of cyclin A1, we searched for transcription factors and cis-regulatory DNA elements. We found that overexpression of PITX2, a paired-like homeodomain transcription factor and a downstream effector of Wnt/ß-catenin signaling, resulted in upregulation of cyclin A1 in HEK293 cells and TPC-1 thyroid cancer cells. On the other hand, PITX2 knockdown in TPC-1 cells caused reduced cyclin A1. Promoter reporter assays with a series of deletion constructs determined that the DNA element from -102 to -96 bp of the cyclin A1 promoter is responsible for PITX2-induced gene expression. The result of chromatin immunoprecipitation revealed the occupancy of PITX2 on the cyclin A1 promoter. Taken together, these findings demonstrate that cyclin A1 is a transcriptional target of PITX2. Consistently, our immunohistochemistry result showed up-regulation of cyclin A1 in human papillary thyroid carcinoma, where overexpressed PITX2 has been endorsed in our recent report. Thus, our study provides new evidence on the regulation of cyclin A1 gene expression and offers a PITX2-cycin A1 pathway for cell cycle regulation.

Dispensable role of PTEN in mouse spermatogenesis.

PTEN (phosphatase and tensin homologue deleted on chromosome ten) plays critical roles in multiple cellular processes, including cell proliferation, survival, migration and transformation. A role of PTEN in mammalian spermatogenesis, however, has not been explored. To address this question, we generated a mouse model with PTEN conditional knockout in postnatal male germ cells. We found that spermatogenesis was normal in PTEN-deleted male germ cells. PTEN conditional mutant males produced sperm and sired offspring as competently as wild-type littermates. Moreover, our biochemical analysis also indicated that the Akt (acutely transforming retrovirus AKT8 in rodent T cell lymphoma) signalling pathway was not affected in mutant testis. Taken together, these findings demonstrate that PTEN is dispensable in mouse spermatogenesis.

Pituitary homeobox 2 (PITX2) promotes thyroid carcinogenesis by activation of cyclin D2.

Pituitary homeobox 2 (PITX2), a Paired-like homeodomain transcription factor and a downstream effector of ß-catenin signaling, plays substantial roles in normal embryonic development but its possible involvement in tumorigenesis was unknown. In this study, we extend its function in human cancer. Remarkably, we found that PITX2 was frequently expressed in human follicular cell-derived (papillary, follicular and anaplastic) thyroid cancer tissues but not in normal thyroids, indicating for the first time that overactivated PITX2 may contribute to thyroid cancer. Cell-based and biochemical studies were performed to uncover the molecular mechanism of PITX2 action in thyroid cancer. Knockdown of PITX2 gene expression in human thyroid cancer cells significantly reduced cell proliferation and soft-agar colony formation. Biochemical analysis of cell cycle regulators upon PITX2 knockdown revealed downregulation of Cyclin D1, Cyclin D2 and dephosphorylation of Rb. Chromatin immunoprecipitation and promoter reporter assay indicated that Cyclin D2 was a direct target gene of PITX2. Consistently, we observed that high expression levels of Cyclin D2 were frequently associated with PITX2 expression in follicular cell-derived thyroid cancer tissues. to confirm our results in vivo, we took advantage of a mouse model of thyroid cancer (TRbeta(PV/PV) mouse). Consistently, the aberrant elevation of PITX2 levels in the thyroid cancer of TRbeta(PV/PV) mice was accompanied by upregulation of Cyclin D1, Cyclin D2 and increased phosphorylation of Rb. Collectively, our findings demonstrate that the overactivated PITX2-Cyclin D2 pathway promotes thyroid tumorigenesis, and they provide the first evidence implicating an oncogenic role of PITX2 in human cancer.

Expression and function of CD9 in melanoma cells.

CD9, a member of the tetraspanin family, functions as an organizer in "tetraspanin webs," through interacting with other cell adhesion molecules. It plays a role in differentiation, fertilization, and cell migration. We investigated the expression and function of CD9 in melanoma. CD9 protein expression in B16 mouse melanoma and six human melanoma cell lines was decreased compared to normal melanocytes. B16F1 clones stably overexpressing CD9 had reduced ability to form colonies in soft agar; however, paradoxically these overexpressing clones had increased ability to invade Matrigel. Similarly, transient overexpression of CD9 in the human metastatic melanoma cell line WM9 dramatically decreased anchorage-independent growth, while transient overexpression of CD9 in the radial growth phase cell line SbCl2 resulted in the gain of Matrigel invasion activity. DNA sequencing of CD9 cDNA from all six human melanoma cell lines did not show deletions, insertions, or mutations. Treatment of all six human melanoma cell lines with the histone deacetylase inhibitor trichostatin A increased CD9 levels. The DNA methylation inhibitor 5-aza-cytidine also increased CD9 protein levels with greater increases seen in cell lines derived from more malignant melanomas.

Testase 1 (ADAM 24) a sperm surface metalloprotease is required for normal fertility in mice.

ADAM family members play important roles in various physiological and pathological processes, for example, fertilization, embryogenesis, neurogenesis, and development of asthma and arthritis (Primakoff and Myles, 2000. Trends Genet 16: 83-87; Edwards et al., 2008. Mol Aspects Med 29: 258-289). We previously reported that testase 1 (ADAM 24) is the first identified metalloprotease present on the surface of mature sperm. To investigate a potential role of testase 1 in fertilization, we generated testase 1 deficient mice. Testase 1 null male mice showed reduced fertility, producing only half the number of offspring when compared to wild-type littermates. In a standard in vitro fertilization assay, we found that sperm lacking testase 1 gave rise to polyspermic fertilization, a phenotypic feature that might contribute to failure of normal embryo development due to polyaneuploidy. Furthermore, in vivo, we found that testase 1 null males produced a higher number of polyspermic embryos at the pronuclear stage. These findings suggest that testase 1 is a sperm plasma membrane component which contributes to the prevention of polyspermy at the level of the oocyte plasma membrane.

hnRNP I inhibits Notch signaling and regulates intestinal epithelial homeostasis in the zebrafish.

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.

The 48-kDa alternative translation isoform of PP2A:B56epsilon is required for Wnt signaling during midbrain-hindbrain boundary formation.

Alternative translation is an underappreciated post-transcriptional regulation mechanism. Although only a small number of genes are found to be alternatively translated, most genes undergoing alternative translation play important roles in tumorigenesis and development. Protein phosphatase 2A (PP2A) is involved in many cellular events during tumorigenesis and development. The specificity, localization, and activity of PP2A are regulated by B regulatory subunits. B56epsilon, a member of the B56 regulatory subunit family, is involved in multiple signaling pathways and regulates a number of developmental processes. Here we report that B56epsilon is alternatively translated, leading to the production of a full-length form and a shorter isoform that lacks the N-terminal 76 amino acid residues of the full-length form. Alternative translation of B56epsilon occurs through a cap-dependent mechanism. We provide evidence that the shorter isoform is required for Wnt signaling and regulates the midbrain/hindbrain boundary formation during Xenopus embryonic development. This demonstrates that the shorter isoform of B56epsilon has important biological functions. Furthermore, we show that the N-terminal sequence of B56epsilon, which is not present in the shorter isoform, contains a nuclear localization signal, whereas the C terminus of B56epsilon contains a nuclear export signal. The shorter isoform, which lacks the N-terminal nuclear localization signal, is restricted to the cytoplasm. In contrast, the full-length form can be localized to the nucleus in a cell type-specific manner. The finding that B56epsilon is alternatively translated adds a new level of regulation to PP2A holoenzymes.

Proteomic and genomic analysis of PITX2 interacting and regulating networks.

Pituitary homeobox 2 (PITX2) is a homeodomain transcription factor that has a substantial role in cell proliferation and differentiation in various tissues. In this report, we have conducted a systematic study, using proteomic and genomic approaches, to characterize PITX2-interacting proteins and PITX2-regulating genes. We identified four novel PITX2-associated protein partners Y box binding factor-1, heterogeneous ribonucleoprotein K, nucleolin and heterogeneous nuclear ribonucleoprotein U in mass spectrometry analysis. We also found that overexpression of PITX2 upregulated 868 genes (2-25-fold) and downregulated 191 genes (2-15-fold) in DNA microarray analysis. These data provide an insightful perspective for further studying PITX2 function and mechanism of action.

Characterization of GPR56 protein and its suppressed expression in human pancreatic cancer cells.

GPR56 is an atypical G protein-coupled receptor (GPCR) with an unusually large N-terminal extracellular region, which contains a long Ser/Thr-rich region forming a mucin-like stalk and due to this feature, GPR56 is thought to be an adhesion GPCR. Recent studies demonstrate that GPR56 plays a role in brain development and tumorigenesis. Here, we report that human GPR56 undergoes GPS (GPCR proteolytic site)-mediated protein cleavage to generate its extracellular domain as an N-terminal fragment (GPR56-N). We also show that GPR56-N is highly glycosylated with N-linked carbohydrate chains. Mouse Gpr56 is ubiquitously expressed in various tissues, with high levels in kidney and pancreas. GPR56 mRNA is detected in diverse human cancer cells including pancreatic cancer cells PANC-1, Capan-1, and MiaCaPa-2. Interestingly, GPR56 protein is either negligible or undetectable in these pancreatic cancer cells, despite the fact that high levels of GPR56 mRNA are observed. Moreover, we have found that protein levels of GPR56 in pancreatic cancer cells were not affected when cells were treated with a proteasome inhibitor MG132. Taken together, these results define the biochemical properties of GPR56 protein, and suggest that the expression of GPR56 protein is suppressed in human pancreatic cancer cells.

Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3.

The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.

Protein kinase C activity in mouse eggs regulates gamete membrane interaction.

Gamete membrane interaction is critical to initiate the development of a new organism. The signaling pathways governing this event, however, are poorly understood. In this report, we provide the first evidence that protein kinase C activity in mouse eggs plays a crucial role in the regulation of this process. Stimulating PKC activity in mouse eggs by phorbol 12-myristate 13-acetate (PMA) drastically inhibited the egg's membrane ability to bind and fuse with sperm. Surprisingly, this significant reduction of gamete membrane interaction was also observed in eggs treated with the PKC inhibitors staurosporine and calphostin c. In further analysis, we found that while no change of egg actin cytoskeleton was detected after either PMA or calphostin c treatment, the structural morphology of egg surface microvilli was severely altered in the PMA-treated eggs, but not in the calphostin c-treated eggs. Moreover, sperm, which bound but did not fuse with the eggs treated with the anti-CD9 antibody KMC8, were liberated from the egg membrane after PMA, but not calphostin c, treatment. Taken together, these results suggest that egg PKC may be precisely balanced to regulate gamete membrane interaction in a biphasic mode, and this biphasic regulation is executed through two different mechanisms.