Niketa A. Patel

Adult Bone Marrow Stromal Cells Differentiate into Neural Cells in Vitro

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.

Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CbetaII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40.

Insulin regulates alternative splicing of PKCβII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648–22654). Transient transfection of constitutively active Akt2 kinase promotes PKCβII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCβII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCβII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCβI and -βII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCβII levels in Akt(-/-), whereas PKCβI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCβII splicing minigene revealed defective βII exon inclusion. Co-transfection of the mutated SRp40 attenuated βII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.

Intravenous Transplants of Human Adipose-Derived Stem Cell Protect the Brain from Traumatic Brain Injury-Induced Neurodegeneration and Motor and Cognitive Impairments: Cell Graft Biodistribution and Soluble Factors in Young and Aged Rats

Traumatic brain injury (TBI) survivors exhibit motor and cognitive symptoms from the primary injury that can become aggravated over time because of secondary cell death. In the present in vivo study, we examined the beneficial effects of human adipose-derived stem cells (hADSCs) in a controlled cortical impact model of mild TBI using young (6 months) and aged (20 months) F344 rats. Animals were transplanted intravenously with 4 × 106 hADSCs (Tx), conditioned media (CM), or vehicle (unconditioned media) at 3 h after TBI. Significant amelioration of motor and cognitive functions was revealed in young, but not aged, Tx and CM groups. Fluorescent imaging in vivo and ex vivo revealed 1,1′ dioactadecyl-3-3-3′,3′-tetramethylindotricarbocyanine iodide-labeled hADSCs in peripheral organs and brain after TBI. Spatiotemporal deposition of hADSCs differed between young and aged rats, most notably reduced migration to the aged spleen. Significant reduction in cortical damage and hippocampal cell loss was observed in both Tx and CM groups in young rats, whereas less neuroprotection was detected in the aged rats and mainly in the Tx group but not the CM group. CM harvested from hADSCs with silencing of either NEAT1 (nuclear enriched abundant transcript 1) or MALAT1 (metastasis associated lung adenocarcinoma transcript 1), long noncoding RNAs (lncRNAs) known to play a role in gene expression, lost the efficacy in our model. Altogether, hADSCs are promising therapeutic cells for TBI, and lncRNAs in the secretome is an important mechanism of cell therapy. Furthermore, hADSCs showed reduced efficacy in aged rats, which may in part result from decreased homing of the cells to the spleen.

Akt2 Regulation of Cdc2-Like Kinases (Clk/Sty), Serine/Arginine-Rich (SR) Protein Phosphorylation, and Insulin-Induced Alternative Splicing of PKCβII Messenger Ribonucleic Acid

Serine/arginine-rich (SR) proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR proteins are regulated by extracellular signals. Regulation of protein kinase C betaII (PKCbetaII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR protein phosphorylation in concert with Akt2. Insulin-stimulated PKCbetaII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR protein phosphorylation, and PKCbetaII expression. Furthermore, we observed that Akt2 phosphorylated several SR proteins distinct from Clk/Sty in response to insulin. Akt2-catalyzed phosphorylation of Clk/Sty and SR proteins revealed a role for both kinases in splicing regulation indicating dual functions for Akt2 in response to insulin in this pathway.

A shift from normal to high glucose levels stimulates cell proliferation in drug sensitive MCF-7 human breast cancer cells but not in multidrug resistant MCF-7/ADR cells which overproduce PKC-βII

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)‐βII in regulating MCF‐7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC‐βII‐specific inhibitor (CGP53353) were examined in cultures of MCF‐7 human breast cancer cell line and its multidrug resistant variant (MCF‐7/ADR). Cell proliferation and DNA synthesis of MCF‐7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF‐7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC‐βII protein and the corresponding mRNA levels were 4‐ to 5‐fold higher in MCF‐7/ADR than in MCF‐7 cells. High glucose‐induced decreases of PKC‐βII protein and mRNA levels were observed during the DNA synthesis phase in MCF‐7 but not in MCF‐7/ADR cells. Decreases in PKC‐βII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose‐stimulated proliferation of MCF‐7 cells. Cultures of multidrug resistant MCF‐7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose‐induced PKC‐βII isozyme down‐regulation with concomitant acceleration of cell cycle progression in MCF‐7 cells. Int. J. Cancer 83:98–106, 1999.

Insulin Regulates Protein Kinase CβII Expression through Enhanced Exon Inclusion in L6 Skeletal Muscle Cells A NOVEL MECHANISM OF INSULIN- AND INSULIN-LIKE GROWTH FACTOR-I-INDUCED 5′ SPLICE SITE SELECTION

The protein kinase Cβ (PKCβ) gene encodes two isoforms, PKCβI and PKCβII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCβ pre-mRNA was examined in L6 myotubes. Mature PKCβII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCβI mRNA and protein levels remained unchanged. Mature PKCβII mRNA resulted from inclusion of the PKCβII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCβII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCβII mRNA, and suggest that PKCβII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5′-splice site variant of PKCβII was also observed. After 30 min, the longer 5′-splice site variant became the predominate species through activation of a downstream 5′ splice site. Similar results were obtained using IGF-I. Although the role of this new PKCβII mRNA species is presently unknown, inclusion of either PKCβII-specific exon results in the same PKCβII protein.

Insulin Promotes Neuronal Survival via the Alternatively Spliced Protein Kinase CδII Isoform

Background: Alternatively spliced PKCδII is a pro-survival protein. Results: Insulin regulates alternative splicing of PKCδII pre-mRNA, which promotes Bcl2 and bcl-xL expression. Conclusion: PKCδII is a key regulator of insulin-mediated neuronal survival. Significance: Elucidation of the molecular mechanisms by which insulin promotes survival in neuronal cells is necessary to understand how intranasal insulin improves cognition. Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells

Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPARγ mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPARγ2 variant was spliced and when terminal differentiation occurs The appearance of PPARγ2 coordinates with the PPARγ1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPARγ1 and, primarily, PPARγ2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPARγ2, but not γ1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPARγ2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events.

PKCδ Alternatively Spliced Isoforms Modulate Cellular Apoptosis in Retinoic Acid-Induced Differentiation of Human NT2 Cells and Mouse Embryonic Stem Cells

NT2 cells are a human teratocarcinoma cell line that, upon treatment with retinoic acid (RA), begin differentiating into a neuronal phenotype. The transformation of undifferentiated NT2 cells into hNT neurons presents an opportunity to investigate the mechanisms involved in neurogenesis because a key component is cell apoptosis, which is essential for building neural networks. Protein kinase Cδ (PKCδ) plays an important role as a mediator of cellular apoptosis in response to various stimuli. PKCδ (δI) is proteolytically cleaved at its hinge region (V3) by caspase 3 and the catalytic fragment is sufficient to induce apoptosis in various cell types. Mouse PKCδII is rendered caspase resistant due to an insertion of 78 bp within the caspase recognition site in its V3 domain. No functional role has been attributed to these alternatively spliced variants of PKCδ. We sought to find a correlation between the onset of apoptosis, neurogenesis, and the expression of PKCδ isoforms. Our results indicate that RA regulates the expression of PKCδ alternative splicing variants in NT2 cells. Further, overexpression of PKCδI promotes apoptosis while PKCδII overexpression shields the cells from apoptosis. This is the first report to attribute physiological function to PKCδI and -δII isoforms. Next we demonstrated that mouse embryonic stem cells differentiate in vitro into dopaminergic neurons upon stimulation with RA and ciliary neurotrophic factor. These cells showed a simultaneous increase in tyrosine hydroxylase and PKCδII expression. We suggest that the molecular mechanisms regulating differentiation and apoptosis could be understood by alternative expression of PKCδ isoforms.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCβ gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCβII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCβI levels were unaltered. PKCβ mRNA levels were depleted by 60–75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCβ expression via the common promoter for PKCβI and PKCβII, deletion constructs of the PKCβ promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (−411 to + 179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5′ promoter region of the PKCβ gene. In actinomycin D‐treated A10 cells, a 60% decrease in PKCβ mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose‐induced posttranscriptional destabilization of PKCβII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to post‐transcriptionally destabilize PKCβII mRNA, but not the PKCβI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.—Patel, N. A., Chalfant, C. E., Yamamoto, M., Watson, J. E., Eichler, D. C., Cooper, D. R. Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells. FASEB J. 13, 103–113 (1999)