Adult muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice.

BACKGROUND: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency. RESULTS: In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1, and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected into streptozotocin (STZ)-treated mice engrafted within 48 h specifically to damaged pancreatic islets and were shown to differentiate and express insulin 10-12 days after injection. In addition, injection of MDSC into hyperglycemic diabetic mice reduced their blood glucose levels for 2-4 weeks. CONCLUSION: These data show that MDSC are capable of differentiating into mature pancreatic beta islet-like cells, not only upon culture in vitro, but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being nonteratogenic, MDSC can be used directly by systemic injection, and this potential reveals a promising alternative avenue in stem cell-based treatment of beta-cell deficiencies.

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase.

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.

Distinct pools of cdc25C are phosphorylated on specific TP sites and differentially localized in human mitotic cells.

BACKGROUND: The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1. METHODOLOGY/PRINCIPAL FINDINGS: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis. CONCLUSIONS/SIGNIFICANCE: These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.

Akt2 is implicated in skeletal muscle differentiation and specifically binds Prohibitin2/REA.

Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.

Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding.

Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

Intracellular retention of caveolin 1 in presenilin-deficient cells.

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy.

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.

Functional cdc25C dual-specificity phosphatase is required for S-phase entry in human cells.

In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.

HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts: implications for vector-dependent plasmid transfection.

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.