Cellular metabolism: Variety is the splice of life

The heterogenous nuclear ribonucleoproteins PTB, hnRNPA1 and hnRNPA2 differentially regulate alternative splicing of pyruvate kinase (PKM) in quiescent and proliferating cells.

The process of aerobic glycolysis, in which cells generate energy by converting glucose to lactate, is unique to proliferating cells. In quiescent cells, glucose is instead metabolized through oxidative phosphorylation. The induction of these discrete metabolic pathways is regulated in part by the alternative splicing of pyruvate kinase (PKM) messenger RNA to generate PKM1, which is expressed in quiescent cells, and PKM2, which is expressed in proliferating cells and induces aerobic glycolysis. In Nature, James Manley and colleagues now report that the production of PKM2 in proliferating cells is regulated by the expression of heterogenous nuclear ribonucleoproteins (hnRNPs).

PKM1 and PKM2 mRNA differ by just one exon: PKM1 contains exon 9, whereas PKM2 contains exon 10. However, it is not known how this alternative splicing is regulated. The mRNA-binding proteins hnRNPA1 and hnRNPA2 were found to interact with specific sequences spanning the exon 9 5' splice site. Furthermore, PTB (polypyrimidine tract binding protein; also called hnRNPI) bound to a site upstream of exon 9. PTB, hnRNPA1 and hnRNPA2 are known to repress splicing, and short interfering (si)RNA against all three proteins, or, in the case of hnRNPA1 and hnRNPA2, mutation of their mRNA binding sites, markedly increased PKM1 mRNA levels in cancer cell lines. Thus, these hnRNP proteins repress generation of PKM1 mRNA.

Intriguingly, hnRNPA1 and PTB levels decreased significantly during the differentiation of the mouse C2C12 cell line. The decrease in hnRNPA1 and PTB correlated with an increase in Pkm1 expression. By contrast, high levels of hnRNPA1, hnRNPA2 and PTB were identified in proliferating glioblastoma multiforme and pilocytic astrocytoma samples; overexpression of PKM2 was also observed in these tissues. The levels of four other splicing factors did not correlate with PKM2 expression, revealing a specific role for hnRNPA1, hnRNPA2 and PTB in this process.

How is the expression of hnRNP proteins upregulated in proliferating cells? The oncogene c-Myc is known to bind to and activate the hnRNPA1, hnRNPA2 and PTB promoters. Depletion of c-Myc in NIH-3T3 fibroblasts not only reduced the accumulation of the hnRNP proteins, but also increased the ratio of Pkm1 to Pkm2. However, this effect was not observed in HeLa cells, suggesting that other transcription factors regulate hnRNP expression in different cell types.

These data reveal that hnRNPA1, hnRNPA2 and PTB are critical regulators of PKM isoform expression. It will be important to determine the precise mechanism by which hnRNP binding promotes exon 10 inclusion — and therefore generation of PKM2 — in proliferating cells.

Emily J. Chenette
Signaling Gateway

Reference:
David, C. J., Chen, M., Assanah, M., Canoll, P. & Manley, J. L.
HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer
Nature advance online publication, 13 December 2009 (DOI 10.1038/nature08697)
Full text | PDF | Subscribe to Nature

Further reading:
Chen, M. & Manley, J. L.
Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches
Nature Reviews Molecular Cell Biology 10, 741-754 (2009)
Full text | PDF | Subscribe to Nature Reviews Molecular Cell Biology

Hedgehog signaling: A pain in the knee

Inhibition of the hedgehog signaling pathway decreases the severity of osteoarthritis.

The degeneration of articular cartilage, which lines the surface of joints, can lead to osteoarthritis. This degeneration process is remarkably similar to the terminal differentiation of growth-plate chondrocytes, suggesting that inhibition of key skeletal differentiation pathways might lessen the severity of this disease. Indeed, Benjamin Alman and colleagues now report in Nature Medicine that inhibition of hedgehog (Hh) signaling can attenuate the development of osteoarthritis.

In chondrocytes, binding of the Indian Hh (Ihh) ligand to the Patched-1 (PTCH1) receptor induces differentiation by relieving inhibition of the seven-transmembrane protein Smoothened (SMO) and permitting activation of the glioma-associated (GLI) transcription factors. GLI proteins then upregulate expression of Hh target genes including GLI1, PTCH1 and HHIP (Hh interacting protein). Alman and colleagues found that these target genes were overexpressed in human osteoarthritic samples, as well as in the articular cartilage from a mouse model of osteoarthritis. In addition, mice with aberrant activation of the Hh pathway showed cartilage degradation and an increased tendency to develop osteoarthritis. The severity of the disease correlated with the degree of Hh pathway activation.

Genetic or pharmacologic inhibition of the Hh pathway in a mouse model of osteoarthritis significantly decreased collagen X deposition, cartilage degradation and osteoarthritis severity. Remarkably, inhibition of Hh in explant cultures of human osteoarthritic cartilage samples blocked expression of key genetic markers of osteoarthritis, including the metalloproteinases ADAMTS5 and MMP13, the transcription factor RUNX2, and COL10A1. RUNX2 was previously shown to be regulated by Hh, and the authors found that Hh induced ADAMTS5 expression via RUNX2.

Intriguingly, Ihh expression in chondrocytes can be induced by abnormal forces or mechanical stresses, but it is not yet known whether such upregulation of Ihh can promote osteoarthritis. Hh pathway inhibitors are currently under investigation as potential anti-cancer therapeutics, and it will be interesting to determine their efficacy in treating osteoarthritis.

Emily J. Chenette
Signaling Gateway

Reference:
Lin, A. C. et al.
Modulating hedgehog signaling can attenuate the severity of osteoarthritis
Nature Medicine 15, 1421-1425 (2009)
Full text | PDF | Subscribe to Nature Medicine

ERK signaling: An integrative approach to cell-fate decisions

ERK-meditated cell-fate decisions are controlled at multiple levels of the Ras–Raf–MEK–ERK signaling cascade.

The serine/threonine kinase ERK elicits different cellular fates depending on the nature of the upstream signal. For example, epidermal growth factor (EGF) induces transient ERK activation and cell proliferation, whereas nerve growth factor (NGF) stimulates sustained ERK activation and differentiation. Regardless of the upstream signal, ERK is activated by a conserved Ras–Raf–MEK signaling cascade, but the mechanisms that funnel distinct upstream activation signals through a single pathway and yet stimulate divergent ERK-mediated responses are not well understood. In Nature Cell Biology, Walter Kolch and colleagues now show that ERK-meditated cell-fate decisions are regulated at multiple levels of the Ras–Raf–MEK–ERK signaling cascade.

To understand how EGF and NGF promote different cellular responses, the authors identified 143 ERK-interacting proteins that were differentially regulated by EGF and NGF. The kinetics of activation and association provided clues as to how cell-fate decisions are regulated by ERK. The Ras GTPase activating protein neurofibromin 1 (NF1), which negatively regulates Ras activation, formed a stable complex with ERK and Ras in the absence of growth factors. NGF treatment caused robust ERK-stimulated phosphorylation of NF1 and prolonged disassociation of the Ras–ERK–NF1 complex, which correlated with sustained Ras activation. In contrast, EGF weakly stimulated NF1 phosphorylation, which led to transient Ras–ERK–NF1 complex disassociation and Ras activation. Short interfering (si)RNA-mediated depletion of NF1 extended Ras and ERK activation and induced partial cellular differentiation following EGF treatment, suggesting that ERK-mediated regulation of NF1 informs cell-fate decisions by differentially regulating Ras pathway activation.

The ERK-interacting protein PEA-15 blocks ERK nuclear import and interferes with ERK-mediated transcriptional regulation. Similar to NF1, the persistence of the ERK–PEA-15 complex was also differentially regulated by growth factors. NGF, but not EGF, induced sustained disassociation of PEA-15 and ERK and facilitated ERK translocation to the nucleus, which is linked to cellular differentiation. Furthermore, siRNA-mediated knockdown of PEA-15 enabled EGF-stimulated differentiation. Thus, PEA-15-mediated regulation of ERK nuclear localization has an important role in promoting proliferation or differentiation.

NGF and EGF also differentially influenced ERK-mediated regulation of transcription factors and co-regulators. Indeed, NGF, but not EGF, caused prolonged ERK-stimulated phosphorylation of the ERF transcription factor. Phosphorylated ERF–ERK complexes accumulated in the cytoplasm — by sequestering ERF in the cytoplasm, ERK relieved ERF's inhibitory effects on transcription and promoted differentiation. In addition, ERK-dependent phosphorylation of the transcriptional regulator TRPS1, which represses the function of GATA transcription factors and hence inhibits cell differentiation, occurred more strongly in response to NGF than EGF. A TRPS1 mutant that could not be phosphorylated blocked differentiation, suggesting that NGF promotes differentiation by stimulating robust TRPS1 phosphorylation.

A computational model of how different signals affect ERK nuclear localization and activity supported the hypothesis that these parameters are regulated through the concerted perturbation of several nodes, rather than one master switch. Thus, the ERK-meditated cell-fate decision is an integrative process controlled at multiple levels of the Ras–Raf–MEK–ERK signaling cascade. It will be important to examine whether this regulatory pathway is conserved in other cell types.

Emily J. Chenette
Signaling Gateway

Reference:
von Kriegsheim, A. et al.
Cell fate decisions are specified by the dynamic ERK interactome
Nature Cell Biology 11, 1458-1464 (2009)
Full text | PDF | Subscribe to Nature Cell Biology