Skeletal abnormalities caused by a Connexin43R239Q mutation in a mouse model for autosomal recessive craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and flaring metaphyses of long bones. Many patients with CMD suffer from neurological symptoms. To date, the pathogenesis of CMD is not fully understood. Treatment is limited to decompression surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a R239Q mutation in CX43. Cx43KI/KI mice replicate many features of AR CMD in craniofacial and long bones. In contrast to Cx43+/+ littermates, Cx43KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones, leading to an expanded bone marrow cavity and increased cortical bone thickness. Although formation of Cx43+/+ and Cx43KI/KI resting OCs are comparable, on bone chips the actively resorbing Cx43KI/KI OCs resorb less bone. Cortical bones of Cx43KI/KI mice have an increase in degenerating osteocytes and empty lacunae. Osteocyte dendrite formation is decreased with reduced expression levels of Fgf23, Sost, Tnf-α, IL-1ß, Esr1, Esr2, and a lower Rankl/Opg ratio. Female Cx43KI/KI mice display a more severe phenotype. Sexual dimorphism in bone becomes more evident as mice age. Our data show that the CX43R239Q mutation results in mislocalization of CX43 protein and impairment of gap junction and hemichannel activity. Different from CX43 ablation mouse models, the CX43R239Q mutation leads to the AR CMD-like phenotype in Cx43KI/KI mice not only by loss-of-function but also via a not yet revealed dominant function.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Generation of Keratinocytes from Human Induced Pluripotent Stem Cells Under Defined Culture Conditions.

Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.

Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia.

Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (AnkKI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in AnkKO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.

BMP signaling-driven osteogenesis is critically dependent on Prdx-1 expression-mediated maintenance of chondrocyte prehypetrophy.

During endochondral ossification, cartilage template is eventually replaced by bone. This process involves several well characterized, stereotypic, molecular and cellular changes in the cartilage primordia. These steps involve transition from resting to proliferative and then pre-hypertrophic to finally hypertrophic cartilage. BMP signaling is necessary and sufficient for osteogenesis. However, the specific step(s) of endochondral ossification in which BMP signaling plays an essential role is not yet known. In this study we have identified Prdx1, a known scavenger of ROS, to be expressed in pre-hypertrophic chondrocytes in a BMP signaling-dependent manner. We demonstrate that BMP signaling inhibition increases ROS levels in osteogenic cells. Further, Prdx1 regulates osteogenesis in vivo by helping maintenance of Ihh expressing pre-hypertrophic cells, in turn regulating these cells' transition into hypertrophy. Therefore, our data suggests that one of the key roles of BMP signaling in endochondral ossification is to maintain pre-hypertrophic state.

Craniometaphyseal Dysplasia Mutations in ANKH Negatively Affect Human Induced Pluripotent Stem Cell Differentiation into Osteoclasts.

We identified osteoclast defects in craniometaphyseal dysplasia (CMD) using an easy-to-use protocol for differentiating osteoclasts from human induced pluripotent stem cells (hiPSCs). CMD is a rare genetic bone disorder, characterized by life-long progressive thickening of craniofacial bones and abnormal shape of long bones. hiPSCs from CMD patients with an in-frame deletion of Phe377 or Ser375 in ANKH are more refractory to in vitro osteoclast differentiation than control hiPSCs. To exclude differentiation effects due to genetic variability, we generated isogenic hiPSCs, which have identical genetic background except for the ANKH mutation. Isogenic hiPSCs with ANKH mutations formed fewer osteoclasts, resorbed less bone, expressed lower levels of osteoclast marker genes, and showed decreased protein levels of ANKH and vacuolar proton pump v-ATP6v0d2. This proof-of-concept study demonstrates that efficient and reproducible differentiation of isogenic hiPSCs into osteoclasts is possible and a promising tool for investigating mechanisms of CMD or other osteoclast-related disorders.

Identification of ASAH1 as a susceptibility gene for familial keloids.

Keloids result from abnormal proliferative scar formation with scar tissue expanding beyond the margin of the original wound and are mostly found in individuals of sub-Saharan African descent. The etiology of keloids has not been resolved but previous studies suggest that keloids are a genetically heterogeneous disorder. Although possible candidate genes have been suggested by genome-wide association studies using common variants, by upregulation in keloids or their involvement in syndromes that include keloid formation, rare coding variants that contribute to susceptibility in non-syndromic keloid formation have not been previously identified. Through analysis of whole-genome data we mapped a locus to chromosome 8p23.3-p21.3 with a statistically significant maximum multipoint LOD score of 4.48. This finding was followed up using exome sequencing and led to the identification of a c.1202T>C (p.(Leu401Pro)) variant in the N-acylsphingosine amidohydrolase (ASAH1) gene that co-segregates with the keloid phenotype in a large Yoruba family. ASAH1 is an acid ceramidase known to be involved in tumor formation by controlling the ratio of ceramide and sphingosine. ASAH1 is also involved in cell proliferation and inflammation, and may affect the development of keloids via multiple mechanisms. Functional studies need to clarify the role of the ASAH1 variant in wound healing.

Genome-editing technologies and patent landscape overview.

Unlike with zinc finger nuclease and transcriptional activator-like effector nuclease DNA modification technologies that rely on lead proteins, developed through expensive and time-consuming processes, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has rapidly emerged as the most promising gene-editing technology to date for the modification of any selected DNA sequence. CRISPR is receiving tremendous fanfare due, in part, to its potential to provide a means to fundamentally alter medical genetics and especially cancer medicine. In this review, we compare key technologies of genome-editing zinc finger nucleases, transcriptional activator-like effector nucleases and CRISPR, with a focus on the race to acquire lucrative intellectual property rights, the current CRISPR patent dispute and potential repercussions on innovation and the adoption of this promising technology by the medical community.

Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation.

Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3ß are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3ß was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3ß was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3ß-dependent manner and thus provides a plausible explanation for GSK3ß-mediated bone loss as described before.

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation.

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3ß), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3ß are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3ß negatively regulates G-CSFR expression and its downstream signaling.

E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha.

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells.

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.

Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes.

Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5-7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone.

Proteomics approaches for myeloid leukemia drug discovery.

INTRODUCTION: Global protein expression profiling between healthy vs diseased states helps identifying differential expression and post-translational modifications of proteins, thereby providing better insights into the molecular changes of disease diagnosis and prognosis. In addition, analytical separation and identification of proteins from complex mixtures can provide insight into targeted drug therapy and prediction of response to different therapeutics. AREAS COVERED: In the present review the authors summarize the readily available quantitative proteomics tools for the analytical separation and identification of target proteins in myeloid leukemia, AML in particular, and its future perspectives in its diagnostics and therapeutics. Within, the authors highlight some of the proteomics approaches such as gel-based quantitation strategies (2D, 2D-DIGE); MS-based quantitative proteomics tools (metabolic labeling (SILAC), chemical labeling (ITRAQ, ICAT)); MS techniques (MALDI-MS/MS). In addition, some of the target proteins identified using proteomics approaches in myeloid leukemia are also discussed that may encourage cancer biology investigators to undertake proteomics as a vital tool in their study. EXPERT OPINION: With suitable, selective application of diverse set of quantitative proteomics strategies integrated with bioinformatics software and precise statistical analysis in myeloid leukemia holds tremendous promise in deciphering cancer proteome, understanding tumor pathophysiology and development of personalized molecular medicine and therapy.

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells.

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.

2-D gel electrophoresis-based proteomic analysis reveals that ormeloxifen induces G0-G1 growth arrest and ERK-mediated apoptosis in chronic myeloid leukemia cells K562.

Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose-dependent manner in a variety of leukemia cells, more strikingly in K562. 2-DE-gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell-cycle pathways, respectively. Our data demonstrate that ormeloxifen-induced apoptosis in K562 cells involves activation of extracellular signal-regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria-mediated caspase-3 activation. Ormeloxifen-induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0-G1 phase by inhibiting c-myc promoter via ormeloxifen-induced MBP-1 (c-myc promoter-binding protein) and upregulation of p21 expression. We further show that ormeloxifen-induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0-G1 phase by reciprocal regulation of p21 and c-myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility.