Ageing: Notching up muscle regeneration

Muscle stem cell proliferation and regeneration is inhibited by an age-associated decrease in active Notch that permits Smad3-mediated transcription of cyclin-dependent kinase inhibitors.

Skeletal muscle regenerates after injury via muscle stem cells known as satellite cells, which re-enter the cell cycle and differentiate into myofibers. Notch signaling stimulates the regenerative capacity of these satellite cells, and the impaired ability of older muscles to recover from injury is associated with the ageing-related decrease in Notch activity. In Nature, Carlson et al. now provide additional mechanistic insight into this process by showing that the transcription factor Smad3 opposes Notch function and blocks myofiber regeneration in old muscles by upregulating the cyclin-dependent kinase inhibitor (CKI) cell cycle regulators.

TGF-β signaling promotes Smad3 phosphorylation (pSmad3), which induces CKIs and cell-cycle arrest. Conversely, Notch signaling is known to repress expression of the CKIs p15 and p21. Carlson et al. noted that aged, injured muscles contained comparatively high levels of TGF-β and pSmad3, but low levels of active Notch. In young, injured muscle cells, exogenous TGF-β upregulated CKI expression, which was blocked by exogenous Notch.

Chromatin immunoprecipitation experiments revealed that pSmad3, active Notch and RNA polymerase II formed a complex on the p15, p16, p21 and p27 CKI gene promoters. Forced activation of Notch reduced the presence of pSmad3-containing complexes at CKI promoters. These data suggest that active Notch blocks pSmad3-mediated CKI expression in young muscles, whereas the ageing-associated reduction in Notch favors the formation of pSmad3 transcription complexes, potentiating CKI transcription and impeding satellite cell proliferation. In vivo analysis of muscle regeneration revealed that old muscles contained fewer satellite-cell-derived myofibers after injury than young muscles. Short hairpin RNA (shRNA)-mediated depletion of Smad3, or expression of an anti-TGF-β neutralizing antibody, increased the regenerative capacity of old muscles and decreased levels of p15 and p21.

Thus, muscle regeneration is regulated by a delicate balance between Notch and pSmad3 transcriptional complexes. Ageing-related repression of Notch tips the balance towards pSmad3-mediated transcription, which upregulates p15 and p21 and inhibits satellite cell proliferation. The mechanism by which TGF-β and Notch levels are regulated as muscles age remains unknown, as TGF-β does not directly inhibit Notch activation, and active Notch does not inhibit TGF-β/pSmad3 production. However, soluble factors secreted by old myofibers upregulated TGF-β production in young satellite cells, and it will be interesting to determine whether the high levels of TGF-β in old muscles are induced by local or systemic factors.

Emily J. Chenette
Signaling Gateway

Original Reference:
Carlson, M. E., Hsu, M. & Conboy, I. M.
Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells
Nature 454, 528-532 (2008)
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Cell migration: PTEN makes neutrophils immune to distraction

The PI(3)K antagonist PTEN promotes efficient neutrophil migration to sites of infection by prioritizing bacterially derived chemoattractant cues and blocking the response to 'distracting' cues from epithelial and immune cells.

To navigate to sites of infection, neutrophils must prioritize a hierarchy of chemoattractant signals. Neutrophils first sense and migrate towards intermediary endothelial- and immune-derived chemokines, but then detect and home in on short-range cues secreted from the pathogen. Furthermore, neutrophils exposed to both an intermediary cue, such as CXC chemokine ligand 2 (CXCL2) or CXCL8, and an end-target cue, such as formyl peptide (fMLP), will preferentially migrate towards the end-target cue, indicating that it is prioritized when both signals are present. However, it is not yet known how neutrophils 'tune out' the intermediary signals. In Nature Immunology, Paul Kubes and colleagues now report that the phosphatidylinositol-3-OH kinase (PI(3)K) antagonist phosphatase and tensin homolog (PTEN) prioritizes end-stage cues by suppressing PI(3,4,5)P₃ accumulation.

Chemotaxis is mediated by discrete PI(3)K and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and previous studies have shown that p38 is essential for end-target signal-mediated migration. Kubes and colleagues found that the presence of fMLP or CXCL8 caused PTEN to localize at the neutrophil trailing membrane, permitting accumulation of PI(3,4,5)P₃ — and hence actin polymerization — at the leading edge. Neutrophils placed between fMLP and CXCL8 activated p38 as they migrated towards fMLP. In these cells, PTEN was distributed around the entire cell membrane, which suppressed PI(3,4,5)P₃ accumulation. Notably, p38 inhibition reverted this diffuse distribution, leading to accumulation of PI(3,4,5)P₃ at the leading edge and migration towards the intermediary cue.

PTEN-deficient mouse neutrophils navigated randomly, rather than directly towards CXCL2. In contrast to wild-type neutrophils, PTEN-deficient neutrophils migrated equally towards CXCL2 and fMLP when placed between opposing cues; PI(3)K inhibition restored the hierarchical migration towards fMLP. To evaluate the possibility that PTEN blocks the 'distracting' CXCL2 signal once neutrophils sense fMLP, neutrophils migrating towards one chemoattractant were exposed to another chemoattractant placed perpendicularly to the direction of migration. Fewer than 5% of wild-type neutrophils turned in response to CXCL2, whereas 25% of PTEN-deficient neutrophils oriented towards CXCL2. Conversely, nearly all wild-type neutrophils migrated towards fMLP when it was used as the second signal, whereas the random migration of PTEN-deficient neutrophils persisted even after addition of fMLP. When examined in vivo, mice with PTEN-deficient neutrophils exhibited a ten-fold higher bacterial burden and prolonged infection due to defective neutrophil recruitment at the site of infection. Thus, PTEN is required to prioritize the end-stage cue, which is essential for an efficient immune response.

These data suggest a model in which neutrophils migrating towards an intermediary cue establish polarized PTEN accumulation away from the leading edge, promoting PI(3)K-mediated migration. End-target chemoattractants activate p38, which causes PTEN to localize around the entire membrane and antagonizes PI(3)K, preventing 'distraction' from intermediary cues. It will be interesting to determine the mechanism by which PTEN is dynamically redistributed, as there is evidence that the p38 signaling pathway may influence PTEN membrane association.

Emily J. Chenette
Signaling Gateway

Original Reference:
Heit, B. et al.
PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils
Nature Immunology 9, 743-752 (2008)
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Billadeau, D. D.
PTEN gives neutrophils direction
Nature Immunology 9, 716-718 (2008)
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Ovarian stem cells: Mei acts like a Brat

Mei-P26 suppresses cell growth and differentiation and downregulates microRNA expression in the Drosophila ovarian stem-cell lineage.

Drosophila neuroblasts and ovarian stem cells divide asymmetrically to create one self-renewing daughter cell and one differentiating daughter cell. In neuroblasts, Brat (brain tumor) — a Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-4 domain) domain-containing protein — segregates into a single daughter cell and inhibits self-renewal. The ovarian stem-cell niche secretes factors that regulate stem-cell proliferation, but a protein with an analogous function to Brat has not yet been identified. In Nature, Neumüller et al. report that the Trim-NHL domain-containing protein Mei-P26 promotes differentiation by regulating microRNA expression in the ovarian stem-cell lineage.

Drosophila oogenesis proceeds from the differentiating daughter cell, which divides to produce 16 cystocytes that develop into an interconnected cyst containing one oocyte and 15 nurse cells. Mei-P26 mRNA and protein expression was low in stem cells, but increased in cyst cells. This selective upregulation of Mei-P26 was dependent on the fusome component Bam (bag of marbles), which is known to be important for cystocyte maturation. Mei-P26-mutant ovaries contained a mass of undifferentiated, abnormally large cystocyte-like cells that proliferated in a tumorous manner. Ectopic expression of Mei-P26 in ovarian stem cells and neuroblasts caused premature differentiation, suggesting a broad role for Trim-NHL domain-containing proteins in stem-cell division and differentiation.

Surprisingly, both Mei-P26 and Brat both bound to Argonaute-1 (AGO1), an RNase component of the RISC (RNA-induced silencing complex). Mature microRNAs were upregulated in the mei-P26 mutant, but diminished following Mei-P26 overexpression. This effect was dependent on an intact NHL domain, as a Mei-P26 mutant that lacked the NHL domain (Mei-P26ΔNHL) failed to bind to AGO1 and could no longer induce differentiation when expressed ectopically. The mei-P26 mutant phenotype in the ovary was partially suppressed by reducing microRNA levels through a heterozygous deletion of the double-stranded RNA binding protein Loquacious. Furthermore, the microRNA bantam, which regulates stem-cell proliferation and apoptosis, was upregulated in mei-P26 mutant ovaries. Exogenous expression of mei-P26, but not mei-P26ΔNHL in cultured Drosophila S2 cells derepressed translation of a bantam target mRNA, establishing a microRNA link between Mei-P26 expression and ovarian stem-cell proliferation.

MicroRNAs are essential for ovarian stem-cell self-renewal, and these data indicate that Mei-P26 modulates differentiation by repressing microRNA expression. The mechanism by which Mei-P26 regulates microRNA biogenesis awaits further study, although Mei-P26 contains a RING finger domain, indicating that it may play a role in protein degradation. The homologous function of Brat and Mei-P26 suggests that Trim-NHL domains may impart a specialized role in stem-cell regulation, but whether these domains function similarly in vertebrates remains to be determined.

Emily J. Chenette
Signaling Gateway

Original Reference:
Neumüller, R. A. et al.
Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage
Nature 454, 241-245 (2008)
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DNA damage: ATM and damage recovery are joined at the HIPK

The ATM and ATR kinases block Siah-1-mediated ubiquitination of HIPK2 during the DNA damage response.

The DNA damage response is regulated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad-3 related). ATM phosphorylates a number of targets involved in the damage response, including p53, which triggers a cell cycle checkpoint and DNA repair or apoptosis depending on the severity of damage. An ATM-dependent pathway also activates homeodomain-interacting protein kinase 2 (HIPK2), which promotes apoptosis by phosphorylating and activating p53. However, the biological mechanism that links DNA damage and ATM/ATR signaling to HIPK2 activation is not yet clear. In Nature Cell Biology, Thomas Hofmann and colleagues now report that ATM phosphorylates the nuclear E3 ubiquitin ligase Siah-1 (seven in absentia homolog-1), preventing Siah-1-mediated ubiquitination and degradation of HIPK2 in response to DNA damage.

HIPK2 is quickly degraded in unstressed cells. Pharmacologic inhibition of the proteasome increased steady-state levels of HIPK2, whereas overexpression of Siah-1 promoted proteasome-mediated degradation of HIPK2. Furthermore, Siah-1 interacted with and ubiquitinated HIPK2 at multiple lysine residues in vitro and in vivo, indicating that HIPK2 is a novel Siah-1 substrate and is regulated by proteasomal degradation.

UV or ionizing radiation-induced DNA damage transiently upregulated HIPK2. However, RNA interference (RNAi)-mediated downregulation of Siah-1 in irradiated cells caused persistent accumulation of HIPK2 and induction of apoptosis. Siah-1 is a p53 target gene, and HIPK2 depletion correlated with p53-induced Siah-1 expression. p53-deficient cells contained high levels of HIPK2 that persisted long after DNA damage.

These results present a conundrum: If p53 mediates HIPK2 degradation through the induction of Siah-1, how is HIPK2 is stabilized in response to DNA damage when p53 is also active? This apparent paradox was resolved upon the observation that the interaction between HIPK2 and Siah-1 is selectively blocked following DNA damage. The authors had previously shown that an ATM-dependent pathway activates HIPK2 following DNA damage, and expression of ATM and ATR protected HIPK2 from Siah-1-medaited degradation. ATM directly phosphorylated Siah-1 at Ser 19 in vivo, and a phosphomimetic Siah-1^S19D mutant did not efficiently interact with HIPK2 in vitro.

These data suggest a model where DNA damage-activated ATM/ATR blocks the Siah-1/HIPK2 interaction, leading to HIPK2 accumulation and hence activation of p53. As cells recover from damage, Siah-1 ubiquitinates HIPK2 and targets it for degradation. It will be interesting to determine how the Siah-1/HIPK2 complexes re-form during recovery, as dephosphorylation or p53-mediated transcription of Siah-1 could both potentially influence this process.

Emily J. Chenette
Signaling Gateway

Original Reference:
Winter, M. et al.
Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR
Nature Cell Biology 10, 812-824 (2008)
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