Ugur Salli

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)‐like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self‐renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.—Mathew, R., Jia, W., Sharma, A., Zhao, Y., Clarke, L. E., Cheng, X., Wang, H., Salli, U., Vrana, K. E., Robertson, G. P., Zhu, J., Wang, S. Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J. 24, 2702–2715 (2010). www.fasebj.org

Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NΔ150 and NΔ150/CΔ24) using isopropyl β-d-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH2-terminal regulatory domain. The solubility of hTPH2, NΔ150, and NΔ150/CΔ24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t½ at 37 °C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NΔ150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH2-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH2-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase Vmax values. These data identify the NH2-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

SelSA, selenium analogs of SAHA as potent histone deacetylase inhibitors.

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC(50) values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.

TPH2 in the ventral tegmental area of the male rat brain.

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain. TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.

Human embryonic and mesenchymal stem cells express different nuclear proteomes.

Human embryonic stem cells (hESCs) are characterized by their immortality and pluripotency. Human mesenchymal stem cells (hMSC), on the other hand, have limited self-renewal and differentiation capabilities. The underlying molecular differences that account for this characteristic self-renewal and plasticity are, however, poorly understood. This study reports a nuclear proteomic analysis of human embryonic and bone marrow-derived mesenchymal stem cells. Our proteomic screen highlighted a 5-fold difference in the expression of Reptin52. We show, using two-dimensional difference gel electrophoresis (2-DIGE), western analysis, and quantitative reverse transcriptase polymerase chain reaction, that Reptin52 is more abundantly expressed in hESC than hMSC. Moreover, we observed differential expression of Pontin52 and beta-catenin-proteins known to interact with Reptin52. This difference in the expression of Reptin52 and Pontin52 (known regulators of beta-catenin) further supports a role for Wnt signaling in stem cell self-renewal and proliferation.

Enhanced nuclear proteomics.

Nuclear proteomics provides an opportunity to examine protein effectors that contribute to cellular phenotype. Both the quality and sensitivity of gel‐based nuclear proteomics are limited, however, by the over‐representation of histones in the protein mixture. These highly charged proteins overshadow rare species and interfere with IEF. A nuclear isolation and protein extraction procedure, tested on human embryonic stem cells, is reported that effectively isolates intact nuclei and then depletes the sample of histones by taking advantage of their ability to form an insoluble complex with DNA at lower pH (even under denaturing conditions). Ubiquitous histones and abundant nuclear actin, are depleted up to 99 ± 0.02 and 42 ± 5%, respectively. This technique greatly improves electrofocusing efficacy and nearly doubles the number of detected protein spots. This approach to nuclear protein isolation for 2‐D PAGE opens the door to better investigation of nuclear protein dynamics.

2-D DIGE identification of differentially expressed heterogeneous nuclear ribonucleoproteins and transcription factors during neural differentiation of human embryonic stem cells

Neural stem cells (NSC) are progenitors that can give rise to all neural lineages. They are found in specific niches of fetal and adult brains and grow in vitro as non‐adherent colonies, the neurospheres. These cells express the intermediate filament nestin, commonly considered an NSC marker. NSC can be derived as neurospheres from human embryonic stem cells (hESC). The mechanisms of cellular programming that hESC undergo during differentiation remain obscure. To investigate the commitment process of hESC during directed neural differentiation, we compared the nuclear proteomes of hESC and hESC‐derived neurospheres. We used 2‐D DIGE to conduct a quantitative comparison of hESC and NSC nuclear proteins and detected 1521 protein spots matched across three gels. Statistical analysis (ANOVA n = 3 with false discovery correction) revealed that only 2.1% of the densitometric signal was significantly changed. The ranges of average ratios varied from 1.2‐ to 11‐fold at a statistically significant p‐value <0.05. MS/MS identified 15 regulated proteins previously shown to be involved in chromatin remodeling, mRNA processing and gene expression regulation. Notably, three members of the heterogeneous nuclear ribonucleoprotein family (AUF‐1, and FBP‐1 and FBP‐2) register a 54, 70 and 99% increased expression, highlighting them as potential markers for NSC in vitro derivation. By contrast, Cpsf‐6 virtually disappears with differentiation with an 11‐fold drop in NSC, highlighting this protein as a novel marker for undifferentiated ESC.

Reptin52 expression during in vitro neural differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) give rise to all somatic cell types, including neural cells such as astrocytes, oligodendrocytes and neurons. Commitment of hESC to a neural fate can be achieved via selection and expansion of developing neural stem cells, which, grown into non-adhering colonies called neurospheres, express nestin, a neurofilament marker. Analysis of hESC and hESC-derived neural stem cell nuclear extracts revealed an increased expression of Reptin52 in neurosphere nuclei. The increase in Reptin52 was evident throughout directed neuronal differentiation as assessed by western blotting, quantitative RT-PCR and immunocytochemistry. Reptin52 serves a pivotal regulatory role in nuclear activities such as transcription regulation and histone modification. In that regard, co-immunoprecipitation experiments showed that binding partners of Reptin52 (Pontin52, beta-catenin and ATF-2) associate with this regulatory protein in hESC-derived neuronal precursors. Moreover, expression of two of these proteins (beta-catenin - the end product of the Wnt signaling pathway - and ATF-2) is coordinately regulated with Reptin52.

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.