Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function.

BACKGROUND: The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it's expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. METHODS: ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). RESULTS: Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. CONCLUSIONS: Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. Video Abstract.

Induction of beige-like adipocyte markers and functions in 3T3-L1 cells by Clk1 and PKCßII inhibitory molecules.

Excessive dietary intake of fat results in its storage in white adipose tissue (WAT). Energy expenditure through lipid oxidation occurs in brown adipose tissue (BAT). Certain WAT depots can undergo a change termed beiging where markers that BAT express are induced. Little is known about signalling pathways inducing beiging. Here, inhibition of a signalling pathway regulating alternative pre-mRNA splicing is involved in adipocyte beiging. Clk1/2/4 kinases regulate splicing by phosphorylating factors that process pre-mRNA. Clk1 inhibition by TG003 results in beige-like adipocytes highly expressing PGC1α and UCP1. SiRNA for Clk1, 2 and 4, demonstrated that Clk1 depletion increased UCP1 and PGC1α expression, whereas Clk2/4 siRNA did not. TG003-treated adipocytes contained fewer lipid droplets, are smaller, and contain more mitochondria, resulting in proton leak increases. Additionally, inhibition of PKCßII activity, a splice variant regulated by Clk1, increased beiging. PGC1α is a substrate for both Clk1 and PKCßII kinases, and we surmised that inhibition of PGC1α phosphorylation resulted in beiging of adipocytes. We show that TG003 binds Clk1 more than Clk2/4 through direct binding, and PGC1α binds to Clk1 at a site close to TG003. Furthermore, we show that TG003 is highly specific for Clk1 across hundreds of kinases in our activity screen. Hence, Clk1 inhibition becomes a target for induction of beige adipocytes.

Long noncoding RNA MALAT1 in exosomes drives regenerative function and modulates inflammation-linked networks following traumatic brain injury.

BACKGROUND: Neuroinflammation is a common therapeutic target for traumatic brain injury (TBI) due to its contribution to delayed secondary cell death and has the potential to occur for years after the initial insult. Exosomes from adipose-derived stem cells (hASCs) containing the long noncoding RNA MALAT1 are a novel, cell-free regenerative approach to long-term recovery after traumatic brain injury (TBI) that have the potential to modulate inflammation at the genomic level. The long noncoding RNA MALAT1 has been shown to be an important component of the secretome of hASCs. METHODS: We isolated exosomes from hASC containing or depleted of MALAT1. The hASC-derived exosomes were then administered intravenously to rats following a mild controlled cortical impact (CCI). We followed the rats with behavior, in vivo imaging, histology, and RNA sequencing (RNA Seq). RESULTS: Using in vivo imaging, we show that exosomes migrate into the spleen within 1 h following administration and enter the brain several hours later following TBI. Significant recovery of function on motor behavior as well as a reduction in cortical brain injury was observed after TBI in rats treated with exosomes. Treatment with either exosomes depleted of MALAT1 or conditioned media depleted of exosomes showed limited regenerative effects, demonstrating the importance of MALAT1 in exosome-mediated recovery. Analysis of the brain and spleen transcriptome using RNA Seq showed MALAT1-dependent modulation of inflammation-related pathways, cell cycle, cell death, and regenerative molecular pathways. Importantly, our data demonstrates that MALAT1 regulates expression of other noncoding RNAs including snoRNAs. CONCLUSION: We demonstrate that MALAT1 in hASC-derived exosomes modulates multiple therapeutic targets, including inflammation, and has tremendous therapeutic potential for treatment of TBI.

Transformer 2ß homolog (Drosophila) (TRA2B) regulates protein kinase C δI (PKCδI) splice variant expression during 3T3L1 preadipocyte cell cycle.

Obesity is characterized by adipocyte hyperplasia and hypertrophy. We previously showed that PKCδ expression is dysregulated in obesity (Carter, G., Apostolatos, A., Patel, R., Mathur, A., Cooper, D., Murr, M., and Patel, N. A. (2013) ISRN Obes. 2013, 161345). Using 3T3L1 preadipocytes, we studied adipogenesis in vitro and showed that expression of PKCδ splice variants, PKCδI and PKCδII, have different expression patterns during adipogenesis (Patel, R., Apostolatos, A., Carter, G., Ajmo, J., Gali, M., Cooper, D. R., You, M., Bisht, K. S., and Patel, N. A. (2013) J. Biol. Chem. 288, 26834-26846). Here, we evaluated the role of PKCδI splice variant during adipogenesis. Our results indicate that PKCδI expression level is high in preadipocytes and decreasing PKCδI accelerated terminal differentiation. Our results indicate that PKCδI is required for mitotic clonal expansion of preadipocytes. We next evaluated the splice factor regulating the expression of PKCδI during 3T3L1 adipogenesis. Our results show TRA2B increased PKCδI expression. To investigate the molecular mechanism, we cloned a heterologous splicing PKCδ minigene and showed that inclusion of PKCδ exon 9 is increased by TRA2B. Using mutagenesis and a RNA-immunoprecipitation assay, we evaluated the binding of Tra2ß on PKCδI exon 9 and show that its association is required for PKCδI splicing. These results provide a better understanding of the role of PKCδI in adipogenesis. Determination of this molecular mechanism of alternative splicing presents a novel therapeutic target in the management of obesity and its co-morbidities.

Protein kinase C δ (PKCδ) splice variants modulate apoptosis pathway in 3T3L1 cells during adipogenesis: identification of PKCδII inhibitor.

Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKCδ), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKCδII splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKCδ alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKCδII splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity.

Insulin promotes neuronal survival via the alternatively spliced protein kinase CδII isoform.

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.

Vitamin A metabolite, all-trans-retinoic acid, mediates alternative splicing of protein kinase C deltaVIII (PKCdeltaVIII) isoform via splicing factor SC35.

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cdelta (PKCdelta), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCdelta, PKCdeltaVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCdeltaVIII via utilization of a downstream 5' splice site of exon 10 on PKCdelta pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCdeltaVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCdeltaVIII alternative splicing. To identify the cis-elements involved in 5' splice site selection we cloned a minigene, which included PKCdelta exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5' splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCdelta minigene promoted selection of 5' splice site II. Mutation of the SC35 binding site in the PKCdelta minigene abolished RA-mediated utilization of 5' splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCdelta exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCdelta pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCdeltaVIII.

Roles of hepatic atypical protein kinase C hyperactivity and hyperinsulinemia in insulin-resistant forms of obesity and type 2 diabetes mellitus.

Diet-induced obesity, the metabolic syndrome, type 2 diabetes (DIO/MetS/T2DM), and their adverse sequelae have reached pandemic levels. In mice, DIO/MetS/T2DM initiation involves diet-dependent increases in lipids that activate hepatic atypical PKC (aPKC) and thereby increase lipogenic enzymes and proinflammatory cytokines. These or other hepatic aberrations, via adverse liver-to-muscle cross talk, rapidly impair postreceptor insulin signaling to glucose transport in muscle. The ensuing hyperinsulinemia further activates hepatic aPKC, which first blocks the ability of Akt to suppress gluconeogenic enzyme expression, and later impairs Akt activation, further increasing hepatic glucose production. Recent findings suggest that hepatic aPKC also increases a proteolytic enzyme that degrades insulin receptors. Fortunately, all hepatic aberrations and muscle impairments are prevented/reversed by inhibition or deficiency of hepatic aPKC. But, in the absence of treatment, hyperinsulinemia induces adverse events, some by using "spare receptors" to bypass receptor defects. Thus, in brain, hyperinsulinemia increases Aß-plaque precursors and Alzheimer risk; in kidney, hyperinsulinemia activates the renin-angiotensin-adrenal axis, thus increasing vasoconstriction, sodium retention, and cardiovascular risk; and in liver, hyperinsulinemia increases lipogenesis, obesity, hepatosteatosis, hyperlipidemia, and cardiovascular risk. In summary, increases in hepatic aPKC are critically required for development of DIO/MetS/T2DM and its adverse sequelae, and therapeutic approaches that limit hepatic aPKC may be particularly effective.

Activation of the nuclear transcription factor SP-1 by insulin rapidly increases the expression of protein kinase C delta in skeletal muscle.

SP-1, a ubiquitous transcription factor involved in regulation of target genes participating in specific signaling pathways, is utilized by insulin for induction of gene transcription. Transcriptional activation generally occurs only after several (14-24) hours. A major element rapidly activated by insulin in skeletal muscle is PKCdelta, which plays a positive regulatory role in insulin signaling. We recently reported that insulin stimulation of skeletal muscle increases PKCdelta RNA expression and PKCdelta protein levels within 5 min. These effects were blocked by inhibitors of either translation or transcription. In this study, we investigated the possibility that SP-1 may participate in this unusually rapid effect. Studies were performed on myoblasts and myotubes of the L6 skeletal muscle cell line. Insulin rapidly increased SP-1 levels and stimulated SP-1 phosphorylation in the nuclear fraction of L6 myotubes. The increase in nuclear SP-1 was blocked by inhibition of nuclear import. Inhibition of SP-1, either pharmacologically or by suppression of SP-1 by RNAi, nearly completely abrogated insulin-induced increase in PKCdelta promoter activity. Insulin induced a rapid association of SP-1 with the PKCalpha promoter. In addition, SP-1 inhibition blocked insulin-induced increases in both PKCdelta RNA expression and PKCdelta protein levels. We conclude that insulin rapidly stimulates SP-1, which mediates the ability of this hormone to induce the rapid transcription of a major target gene utilized in the insulin signaling cascade.

Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing.

Objective: Chronically ill patients heal recalcitrant ulcerative wounds more slowly. Human adipose-derived stem cells (hADSCs) play an important role in tissue regeneration and exosomes secreted by hADSC contribute to their paracrine signaling. In addition to cytokines, lipids and growth factors, hADSC secrete mRNA, miRNA, and long noncoding (lnc) RNA into exosomes. In this study we examined the role of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), an abundant lncRNA in exosomes from conditioned media (CM), on cell migration and ischemic wound healing. Approach: CM and isolated exosomes from hADSC were applied to human dermal fibroblast (HDF) in scratch assays and electric cell-substrate impedance sensing (ECIS) assays. CM was also applied to a rat model of ischemic wound healing and wound closure was followed. Results: CM stimulated cell migration of HDFs in vitro by 48%. CM stimulated the closure of ischemic wounds in a rat model 50% faster than unconditioned media. The depletion of MALAT1 in adipose-derived stem cell (ADSC) CM significantly reduced cell migration. Since MALAT1 is secreted into exosomes, a purified population of exosomes was applied to HDF where they enhanced cell migration in a similar manner to FGF-2 or basic fibroblast growth factor (bFGF) in ECIS wound healing assays. The uptake of exosomes by HDF was shown using dynasore, an inhibitor that blocks clathrin- and caveolin-dependent endocytosis. Depletion of MALAT1 in hADSC with antisense oligonucleotides resulted in exosomes without MALAT1. These exosomes had an effect similar to the unconditioned, control media in ECIS assays. Innovation: Exosomes contain lncRNA MALAT1 and other factors that have the potential to stimulate HDF cell migration and angiogenesis involved in wound healing without applying stem cells to wounds. Conclusion: Our results show the potential of using topically applied ADSC-derived exosomes containing MALAT1 for treating ischemic wounds. This allows for harnessing the power of stem cell paracrine signaling capabilities without applying the cells.

Ceramide-activated protein phosphatase involvement in insulin resistance via Akt, serine/arginine-rich protein 40, and ribonucleic acid splicing in L6 skeletal muscle cells.

Elevated TNFalpha levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of protein kinase CbetaII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFalpha attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase. Western blot analysis of L6 cell lysates demonstrated impaired insulin-stimulated phosphorylation of Akt, serine/arginine-rich protein 40, and glycogen synthase kinase 3beta in response to TNFalpha and the short chain C6 ceramide analog. TNFalpha increased serine/threonine phosphatase activity of protein phosphatase 1 (PP1) in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by liquid chromatography-mass spectrometry showed consistent increases in C24:1 and C16 ceramides. Effects of TNFalpha and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3beta were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFalpha abolished insulin-mediated inclusion of the protein kinase CbetaII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFalpha effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.

Insulin rapidly upregulates protein kinase Cdelta gene expression in skeletal muscle.

Recent studies in our laboratories have shown that Protein Kinase C delta (PKCdelta) is essential for insulin-induced glucose transport in skeletal muscle, and that insulin rapidly stimulates PKCdelta activity skeletal muscle. The purpose of this study was to examine mechanisms of regulation of PKCdelta protein availability. Studies were done on several models of mammalian skeletal muscle and utilized whole cell lysates of differentiated myotubes. PKCdelta protein levels were determined by Western blotting techniques, and PKCdelta RNA levels were determined by Northern blotting, RT-PCR and Real-Time RT-PCR. Insulin stimulation increased PKCdelta protein levels in whole cell lysates. This effect was not due to an inhibition by insulin of the rate of PKCdelta protein degradation. Insulin also increased 35S-methionine incorporation into PKCdelta within 5-15 min. Pretreatment of cells with transcription or translation inhibitors abrogated the insulin-induced increase in PKCdelta protein levels. We also found that insulin rapidly increased the level of PKCdelta RNA, an effect abolished by inhibitors of transcription. The insulin-induced increase in PKCdelta expression was not reduced by inhibition of either PI3 Kinase or MAP kinase, indicating that these signaling mechanisms are not involved, consistent with insulin activation of PKCdelta. Studies on cells transfected with the PKCdelta promoter demonstrate that insulin activated the promoter within 5 min. This study indicates that the expression of PKCdelta may be regulated in a rapid manner during the course of insulin action in skeletal muscle and raise the possibility that PKCdelta may be an immediate early response gene activated by insulin.

MALAT1 in Human Adipose Stem Cells Modulates Survival and Alternative Splicing of PKCδII in HT22 Cells.

Brain injury may be caused by trauma or may occur in stroke and neurodegenerative diseases. Because the central nervous system is unable to regenerate efficiently, there is utmost interest in the use of stem cells to promote neuronal survival. Of interest here are human adipose-derived stem cells (hASCs), which secrete factors that enhance regeneration and survival of neurons in sites of injury. We evaluated the effect of hASC secretome on immortalized mouse hippocampal cell line (HT22) after injury. Protein kinase C δ (PKCδ) activates survival and proliferation in neurons and is implicated in memory. We previously showed that alternatively spliced PKCδII enhances neuronal survival via B-cell lymphoma 2 Bcl2 in HT22 neuronal cells. Our results demonstrate that following injury, treatment with exosomes from the hASC secretome increases expression of PKCδII in HT22 cells and increases neuronal survival and proliferation. Specifically, we demonstrate that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA contained in the hASC exosomes mediates PKCδII splicing, thereby increasing neuronal survival. Using antisense oligonucleotides for MALAT1 and RNA immunoprecipitation assays, we demonstrate that MALAT1 recruits splice factor serine-arginine-rich splice factor 2 (SRSF2) to promote alternative splicing of PKCδII. Finally, we evaluated the role of insulin in enhancing hASC-mediated neuronal survival and demonstrated that insulin treatment dramatically increases the association of MALAT1 and SRSF2 and substantially increases survival and proliferation after injury in HT22 cells. In conclusion, we demonstrate the mechanism of action of hASC exosomes in increasing neuronal survival. This effect of hASC exosomes to promote wound healing can be further enhanced by insulin treatment in HT22 cells.

PKCdelta 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 Cdelta (PKCdelta) plays an important role as a mediator of cellular apoptosis in response to various stimuli. PKCdelta (deltaI) 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 PKCdeltaII 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 PKCdelta. We sought to find a correlation between the onset of apoptosis, neurogenesis, and the expression of PKCdelta isoforms. Our results indicate that RA regulates the expression of PKCdelta alternative splicing variants in NT2 cells. Further, overexpression of PKCdeltaI promotes apoptosis while PKCdeltaII overexpression shields the cells from apoptosis. This is the first report to attribute physiological function to PKCdeltaI and -deltaII 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 PKCdeltaII expression. We suggest that the molecular mechanisms regulating differentiation and apoptosis could be understood by alternative expression of PKCdelta isoforms.

Adipose-derived stem cells from lean and obese humans show depot specific differences in their stem cell markers, exosome contents and senescence: role of protein kinase C delta (PKCδ) in adipose stem cell niche.

BACKGROUND: Adipose-derived stem cells (ASC) and its exosomes are gaining utmost importance in the field of regenerative medicine. The ASCs tested for their potential in wound healing are predominantly derived from the subcutaneous depot of lean donors. However, it is important to characterize the ASC derived from different adipose depots as these depots have clinically distinct roles. METHODS: We characterized the ASC derived from subcutaneous and omental depots from a lean donor (sc-ASCn and om-ASCn) and compared it to the ASC derived from an obese donor (sc-ASCo and om-ASCo) using flow cytometry and real time qPCR. RESULTS: We show that stem cell markers Oct4, Sal4, Sox15, KLF4 and BMI1 have distinct expression patterns in each ASC. We evaluated the secretome of the ASC and characterized their secreted exosomes. We show long noncoding RNAs (lncRNAs) are secreted by ASC and their expression varied between the ASC's derived from different depots. Protein kinase C delta (PKCδ) regulates the mitogenic signals in stem cells. We evaluated the effect of silencing PKCδ in sc-ASCn, om-ASCn, sc-ASCo and om-ASCo. Using ß-galactosidase staining, we evaluated the percentage of senescent cells in sc-ASCn, om-ASCn, sc-ASCo and om-ASCo. Our results also indicated that silencing PKCδ increases the percentage of senescent cells. CONCLUSIONS: Our case-specific study demonstrates a role of PKCδ in maintaining the adipose stem cell niche and importantly demonstrates depot-specific differences in adipose stem cells and their exosome content.

Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression.

Glucose concentration may be an important factor in breast cancer cell proliferation, and the prevalence of breast cancer is high in diabetic patients. Leptin may also be an important factor since plasma levels of leptin correlated with TNM staging for breast cancer patients. The effects of glucose and leptin on breast cancer cell proliferation were evaluated by examining cell doubling time, DNA synthesis, levels of cell cycle related proteins, protein kinase C (PKC) isozyme expression, and peroxisome proliferator-activated receptor (PPAR) subtypes were determined following glucose exposure at normal (5.5 mM) and high (25 mM) concentrations with/without leptin in MCF-7 human breast cancer cells. In MCF-7 cells, leptin and high glucose stimulated cell proliferation as demonstrated by the increases in DNA synthesis and expression of cdk2 and cyclin D1. PKC-alpha, PPARgamma, and PPARalpha protein levels were up-regulated following leptin and high glucose treatment in drug-sensitive MCF-7 cells. However, there was no significant effect of leptin and high glucose on cell proliferation, DNA synthesis, levels of cell cycle proteins, PKC isozymes, or PPAR subtypes in multidrug-resistant human breast cancer NCI/ADR-RES cells. These results suggested that hyperglycemia and hyperleptinemia increase breast cancer cell proliferation through accelerated cell cycle progression with up-regulation of cdk2 and cyclin D1 levels. This suggests the involvement of PKC-alpha, PPARalpha, and PPARgamma.

Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.

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.

Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells.

BACKGROUND: Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject. METHODS: We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student's t-test. RESULTS: We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/ß+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression. CONCLUSIONS: Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC.

Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.

Objective: Adipose tissue is a robust source of adipose-derived stem cells (ADSCs) that may be able to provide secreted factors that promote the ability of wounded tissue to heal. However, adipocytes also have the potential to dedifferentiate in culture to cells with stem cell-like properties that may improve their behavior and functionality for certain applications. Approach: ADSCs are adult mesenchymal stem cells that are cultured from the stromal vascular fraction of adipose tissue. However, adipocytes are capable of dedifferentiating into cells with stem cell properties. In this case study, we compare ADSC and dedifferentiated fat (DFAT) cells from the same patient and fat depot for mesenchymal cell markers, embryonic stem cell markers, ability to differentiate to adipocytes and osteoblasts, senescence and telomerase levels, and ability of conditioned media (CM) to stimulate migration of human dermal fibroblasts (HDFs). Innovation and Conclusions: ADSCs and DFAT cells displayed identical levels of CD90, CD44, CD105, and were CD34- and CD45-negative. They also expressed similar levels of Oct4, BMI1, KLF4, and SALL4. DFAT cells, however, showed higher efficiency in adipogenic and osteogenic capacity. Telomerase levels of DFAT cells were double those of ADSCs, and senescence declined in DFAT cells. CM from both cell types altered the migration of fibroblasts. Despite reports of ADSCs from a number of human depots, there have been no comparisons of the ability of dedifferentiated DFAT cells from the same donor and depot to differentiate or modulate migration of HDFs. Since ADSCs were from an obese diabetic donor, reprogramming of DFAT cells may help improve a patient's cells for regenerative medicine applications.