Denise R. Cooper

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.

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.

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.

The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8–10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.

Aims/hypothesis Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCβII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements. Methods We examined phosphoPKCβII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCβII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCβII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCβII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353. Results Insulin increased phosphoPKCβII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCβII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells. Conclusions/interpretation The results suggest that PKCβII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Elevated TNFα 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 CβII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFα 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 3β in response to TNFα and the short chain C6 ceramide analog. TNFα 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 TNFα and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3β were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFα abolished insulin-mediated inclusion of the protein kinase CβII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFα effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.

Alternative splicing of pre-mRNA encoding the carboxy-terminal (C-terminal) exons of protein kinase C beta (PKC beta) leads to the expression of two protein isoforms, PKC beta 1 and PKC beta II, with the potential for different functions. PKC beta II expression is regulated by insulin via alternative mRNA splicing. A physiological consequence of its activation was investigated in L6 rat skeletal muscle cells expressing GLUT4 transporters, a cell line in which PKC is involved in glucose transport. We examined the contribution of PKC beta II for insulin-stimulated [3H]2-deoxyglucose uptake by constructing three PKC beta II C-terminal deletion mutants designated M216, M217, and M218. When transiently expressed in COS1 cells, M217, with nine amino acids deleted, demonstrated autophosphorylation activity 10-fold less than full-length PKC beta II. The mutants M218, with 13 amino acids deleted, and M216, with 52 amino acids deleted, demonstrated no autophosphorylation activity and are kinase negative. When transiently expressed in L6 myotubes, M217 inhibited insulin-stimulated 2-deoxyglucose uptake by 45% (with a 45% transfection efficiency) whereas M216 and M218, kinase-negative mutants, had no effect compared with cells transfected with control plasmid. Cotransfection of full-length PKC beta II with M217 was able to rescue the inhibition of insulin-stimulated 2-deoxyglucose uptake as compared with cotransfection of M217 with the control plasmid, suggesting that M217 acts as a dominant-negative. In contrast, cotransfection of full-length PKC beta I, the other alternatively spliced form, did not rescue inhibition of insulin-stimulated 2-deoxyglucose uptake by M217. To further demonstrate the involvement of PKC, specifically PKC beta II, in insulin-stimulated 2-deoxyglucose uptake, we used two inhibitors, CG41251 (a specific PKC inhibitor) and CG53353 (a PKC beta II-specific inhibitor at 1 microM). Both inhibited insulin-stimulated 2-deoxyglucose uptake 50-60% in L6 myotubes. We conclude that M217 may act as a specific PKC beta II dominant-negative and that PKC beta II is more specific for insulin-stimulated 2-deoxyglucose uptake in these cells than PKC beta I.