Am J Physiol Cell Physiol 306: C762–C767, 2014. First published February 19, 2014; doi:10.1152/ajpcell.00361.2013.

NF-␬B but not FoxO sites in the MuRF1 promoter are required for transcriptional activation in disuse muscle atrophy Chia-Ling Wu, Evangeline W. Cornwell, Robert W. Jackman, and Susan C. Kandarian Department of Health Sciences, Boston University, Boston, Massachusetts Submitted 26 November 2013; accepted in final form 14 February 2014

Wu CL, Cornwell EW, Jackman RW, Kandarian SC. NF-␬B but not FoxO sites in the MuRF1 promoter are required for transcriptional activation in disuse muscle atrophy. Am J Physiol Cell Physiol 306: C762–C767, 2014. First published February 19, 2014; doi:10.1152/ajpcell.00361.2013.—The muscle-specific ring finger protein 1 (MuRF1) gene is required for most types of skeletal muscle atrophy yet we have little understanding of its transcriptional regulation. The purpose of this study is to identify whether NF-␬B and/or FoxO response elements in the MuRF1 promoter are required for MuRF1 gene activation during skeletal muscle atrophy due to the removal of hindlimb weight bearing (“unloading”). Both NF-␬B -dependent and FoxO-dependent luciferase reporter activities were significantly increased at 5 days of unloading. Using a 4.4-kb MuRF1 promoter reporter construct, a fourfold increase in reporter (i.e., luciferase) activity was found in rat soleus muscles after 5 days of hindlimb unloading. This activation was abolished by mutagenesis of either of the two distal putative NF-␬B sites or all three putative NF-␬B sites but not by mutagenesis of all four putative FoxO sites. This work provides the first direct evidence that NF-␬B sites, but not FoxO sites, are required for MuRF1 promoter activation in muscle disuse atrophy in vivo. NF-␬B; FoxO; MuRF1; muscle wasting; unloading THE MUSCLE-SPECIFIC RING FINGER protein 1 (MuRF1 or Trim63) is an E3 ubiquitin ligase that has been shown to be a key regulator of protein degradation during skeletal muscle atrophy. Several studies indicate that MuRF1 ubiquitinates sarcomeric proteins such as myosin heavy chain (6), myosin binding protein C, and myosin light chain 1 and 2 for proteasomal degradation during muscle atrophy (7). Others have shown that MuRF1 protein regulates energy metabolism and inhibits protein synthesis during skeletal muscle atrophy (1, 10, 16). Importantly, knockout of the MuRF1 gene attenuates muscle atrophy in response to denervation (2), glucocorticoid treatment (1), and hindlimb unloading (17). MuRF1 expression is increased in muscle atrophy due to a variety of physiological triggers including hindlimb unloading (2, 15, 29, 33), denervation (2), limb immobilization (2, 28), starvation (18), glucocorticoid administration (32), acute lung injury (9), and cancer cachexia (3, 18, 23). Despite the number of studies showing an important role of MuRF1 in skeletal muscle atrophy, our understanding of MuRF1 gene regulation is incomplete. MuRF1 upregulation in denervated muscle was blocked in myogenin knockout mice, and, mutagenesis of myogenin binding sites in a 600-bp MuRF1 promoter construct blocked the MuRF1 reporter activation due to denervation (21). In other work, putative FoxO binding sites and glucocorticoid response elements were required for activation of a 500-bp MuRF1 promoter due to

Address for reprint requests and other correspondence: S. C. Kandarian, Dept. of Health Sciences, Boston Univ., 635 Commonwealth Ave., Boston, MA 02215 (e-mail: [email protected]). C762

glucocorticoid treatment of HepG2 cells (32). In muscle atrophy due to acute lung injury, a 5-kb MuRF1 promoter containing multiple NF-␬B sites was strongly activated whereas the proximal 500-bp MuRF1 promoter without the ␬B sites was not activated (9). We recently found that deletion of 2.3 kb (containing all putative ␬B sites) of the 5=-end of a MuRF1 promoter construct abolished unloading inducibility (14). This experiment demonstrated that the two most proximal FoxO sites and all the Ebox (myogenin) binding sites do not participate in the induction of the MuRF1 gene by unloading. These data suggest that a distal region of the MuRF1 promoter containing NF-␬B and FoxO binding sites may be involved in MuRF1 gene regulation during unloading atrophy but primary evidence is lacking. Thus there is a need for a more complete analysis of the MuRF1 promoter using site-directed mutagenesis of longer constructs than those previously used. In addition, in vivo analysis of site-specific mutant constructs is needed to determine the role of specific transcription factors in the regulation of physiological muscle atrophy. In the case of muscle disuse FoxO and NF-␬B transcription factors are required for atrophy in vivo (12, 24, 33). The purpose of the present study is to determine if the putative FoxO and NF-␬B sites of the MuRF1 promoter (4.4 kb) are required for the transactivation of MuRF1 during disuse by the removal of weight bearing, a clinically relevant model of skeletal muscle atrophy. This work provides primary evidence that NF-␬B sites, but not FoxO sites, are required for MuRF1 upregulation during disuse atrophy, supporting the idea that different transcription factors are involved in MuRF1 gene regulation depending on the trigger of muscle atrophy. MATERIALS AND METHODS

Animals and hindlimb unloading. Female Wistar rats, 7 wk of age (Charles River Laboratories, Wilmington, MA), were provided with chow and water ad libitum and were housed individually in the Boston University Animal Care Facility. Animals were used for experiments after 3 days of acclimatization. Disuse muscle atrophy was induced by removing hindlimb weight bearing of rats for 5 days as described previously (26). The use of animals in this study was approved by the Institutional Animal Care and Use Committee of Boston University (Protocol No. 12– 012). Reporter constructs. The NF-␬B-dependent reporter has been described in detail (13). The FoxO-dependent reporter plasmid (a gift from M. Greenberg) containing three Forkhead-responsive elements (3xFHRE) has been previously described (4). The wild-type MuRF1 promoter reporter plasmid contains 4.4 kb of the 5=-promoter region of mouse MuRF1 driving a luciferase reporter gene [a gift from S. Shoelson (5)]. Whole Genome RVista algorithm (http://genome. lbl.gov/vista/citeus.shtml) (20) was used to identify putative transcription factor binding sites conserved between mouse and human genomes. Site-directed mutagenesis of putative NF-␬B and FoxO sites was performed on the 4.4-kb MuRF1 promoter construct using the

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Table 1. Sequence of wild-type and mutant sites of the MuRF1 promoter Sites/Sequences

NF-␬B sites Wild type ␬B mutants

␬B1 (⫺4.2 kb) gaaaagtccc gaaaagtGAG

␬B2 (⫺4.1 kb) gggaagttcc CTCaagttcc

␬B3 (⫺3.1 kb) gggaacttcccc gggaacttGAGc FoxO sites

Wild type FoxO mutants

FBE1 (⫺3.0 kb) gaagtcaacaggaa gaagtcaCcCggaa

FBE2 (⫺2.5 kb) ttgtttcaaa ttgGtGcaaa

FBE3 (⫺185 bp) ttcttgtttacgac ttcttgGtGacgac

FBE4 (⫺24 bp) tataaatatcag tatCaCtatcag

Capital letters are the mutated nucleic acids. MuRF1, muscle-specific ring finger protein 1.

QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s protocols. The mutated primers were designed using the QuikChange Primer Design Program (Agilent Technologies), and then they were synthesized by Invitrogen (Carlsbad, CA). The three NF-␬B sites in the MuRF1 promoter (relative to TSS) were mutated as follows, with the NF-␬B site underlined and mutated nucleotides capitalized: ␬B1 site (⫺4.2 kb): 5=-caa act ctc agg ttt ctg aaa agt GAG ttt tct agt gac aat ccc aaa gag-3=; ␬B2 site (⫺4.1 kb): 5=- ccc aaa gag cac aga ctt aCT Caa gtt cca gcg cta cca g-3=; and ␬B3 site (⫺3.1 kb): 5=-ccg ccc atg tgg gaa ctt GAG cat ctc acc ctt tga ctt-3=. An “all␬Bmut” MuRF1 construct containing all three mutated NF-␬B sites was made by sequentially mutating the NF-␬B sites in the 4.4 kb MuRF1 promoter using the primers described above and was previously described (14). For comparison to wild-type sequences, see Table 1. Mutations of the nucleotides indicated in the putative NF-␬B binding elements have been shown to abolish NF-␬B binding (11). An “allFoxOmut” MuRF1 promoter construct containing four mutated FoxO sites was made by sequentially mutating the putative FoxO binding sites in the wild-type MuRF1 promoter. The FoxO sites are underlined and mutated nucleotides are capitalized (site position relative to TSS): FBE1 site (⫺3.0 kb): 5=-cca gtg tgg ctg agg aag tca CcC gga agc tgt ga-3=; FBE2 site (⫺2.5 kb): 5=-ccg aaa tca cca tgc cct ttg GtG caa aga cct cct tct ata atc-3=; FBE3 site (⫺185 bp): 5=-tga aca gtc tgt tct tgG tGa cga ccc cca cgg cag-3=; and FBE4 site (⫺24 bp): 5=-tga cag agg tgc agc tat CaC tat cag agg ggc ctc ag-3=. For a comparison to wild-type FoxO sequences, see Table 1. Mutations of the nucleotides indicated in the putative forkhead binding elements have been shown to abolish Foxo binding (8). To make the mutant clones, a reaction was performed by mixing 100 ng of each phosphorylated primer, 100 ng 4.4-kb MuRF1 promoter luciferase plasmid, 1.25 U Pfu Ultra High-fidelity DNA polymerase (Agilent), and 20 U Taq DNA ligase (New England Biolabs, Ipswich, MA) and then subjected to thermal cycling as follows: 95°C for 2 min, then 30 cycles of 95°C for 50 s, 60°C for 50 s, and 68°C for 5 min, and followed by a final incubation at 68°C for 5 min. After DpnI treatment, amplified PCR products were transformed into XL10Gold Ultracompetent bacteria according to manufacturer’s instructions (Agilent Technologies). The sequences of wild-type and mutated MuRF1 promoter constructs were verified by Genewiz sequencing (South Plainfield, NJ). In vivo plasmid electrotransfer and reporter assays. In vivo plasmid electrotransfer was performed by injecting 40 ␮g of NF-␬B reporter, FoxO reporter, wild-type MuRF1 reporter, or mutant MuRF1 reporter plasmid into rat soleus muscle followed by five direct pulses at 100 V/cm, 20 ms, 1 Hz, with 200-ms interpulse interval as previously described (31). Twenty-four hours after plasmid injection, half the rats remained weight bearing and half were hindlimb unloaded. Five days later, soleus muscles transfected with reporter plasmid were surgically dissected, snap-frozen in liquid nitrogen, and saved at ⫺80°C. Frozen muscles were mechanically homogenized in 1.5 ml passive lysis buffer (Promega, Madison, WI) on ice. Luciferase

activity was measured as previously described (31) using a TD-20/20 luminometer (Turner Designs). Statistics. For weight-bearing and hindlimb unloaded groups, a Student’s t-test was performed to determine if there were statistically different changes due to the unloading condition. A t-test was performed on muscle mass, body mass, and luciferase activity (weight bearing vs. hindlimb unloading) for each of the six plasmid MuRF1 constructs and for NF-␬B- and FoxO-dependent reporter constructs using GraphPad Prism 5.03 (San Diego, CA). Data are presented as means ⫾ SE, and the number of muscles per group was 8 –10. Statistical significance was set at P ⬍ 0.05 for all t-tests. RESULTS

For each group of rats, we confirmed that 5 days of hindlimb unloading induced soleus muscle atrophy (26 –34%) without a change in body mass (Table 2). Although NF-␬B-dependent reporter activity is significantly increased in soleus muscles due to 3, 7, and 10 days of hindlimb unloading (12, 13, 15, 31) and due to 3 and 7 days of immobilization (28), here we confirm that NF-␬B reporter activity is also elevated at 5 days of unloading (Fig. 1, right). Table 2. Body mass, soleus muscle mass, and muscle massto-body mass ratio for weight-bearing and unloaded groups transfected with either wild-type or mutant MuRF1 promoter plasmids Constructs

Body mass, g MuRF1 (WT) allkBmut MuRF1 allFoxomut MuRF1 kB1mut MuRF1 kB2mut MuRF1 kB3mut MuRF1 Muscle mass, mg MuRF1 (WT) allkBmut MuRF1 allFoxomut MuRF1 kB1mut MuRF1 kB2mut MuRF1 kB3mut MuRF1 Muscle mass/body mass, mg/g MuRF1 (WT) allkBmut MuRF1 allFoxomut MuRF1 kB1mut MuRF1 kB2mut MuRF1 kB3mut MuRF1

Weight Bearing

Hindlimb Unloaded

P Value

185.6 ⫾ 5.38 185.9 ⫾ 4.34 174.8 ⫾ 4.16 196.0 ⫾ 2.04 194.4 ⫾ 2.13 193.0 ⫾ 6.49

190.9 ⫾ 3.09 184.3 ⫾ 4.34 160.8 ⫾ 1.04 186.9 ⫾ 2.10 190.2 ⫾ 2.11 193.2 ⫾ 3.24

NS NS NS NS NS NS

108.4 ⫾ 3.11 115.4 ⫾ 2.29 93.8 ⫾ 4.00 106.2 ⫾ 1.58 107.5 ⫾ 4.70 110.7 ⫾ 1.97

80.2 ⫾ 1.98 85.2 ⫾ 4.22 69.6 ⫾ 3.09 73.1 ⫾ 1.87 70.9 ⫾ 2.12 80.1 ⫾ 2.35

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

0.594 ⫾ 0.026 0.623 ⫾ 0.027 0.536 ⫾ 0.011 0.542 ⫾ 0.008 0.553 ⫾ 0.022 0.573 ⫾ 0.035

0.420 ⫾ 0.012 0.462 ⫾ 0.022 0.433 ⫾ 0.019 0.391 ⫾ 0.008 0.373 ⫾ 0.011 0.414 ⫾ 0.012

⬍0.001 ⬍0.01 ⬍0.01 ⬍0.001 ⬍0.001 ⬍0.001

Values are means ⫾ SE. WT, wild type.

AJP-Cell Physiol • doi:10.1152/ajpcell.00361.2013 • www.ajpcell.org

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Luciferase activity (Fold Change)

5

Weight Bearing Unloading

*

4

3

*

2

1

0

FoxOluc

NF-κBluc

Fig. 1. (3x)-NF-␬B-dependent and (3x)-FoxO-dependent reporter (i.e., luciferase) gene activation in rat soleus muscles due to 5 days of hindlimb unloading. Right: NF-␬B-dependent reporter gene activation in unloaded muscle compared with weight-bearing muscle. Left: FoxO-dependent reporter gene activation in unloaded muscle compared with weight-bearing muscle. *Statistically different from weight-bearing (P ⬍ 0.05).

FoxO-dependent reporter activity was significantly increased in soleus muscles at 3 and 7 days of immobilization (28), and we show that it is also upregulated in soleus muscles after 5 days of unloading (Fig. 1, left). To identify regions of conservation, 4.4 kb of the MuRF1 promoter sequence from mouse and human were aligned and compared (Fig. 2). Three regions in the MuRF1 promoter showed ⬎75% sequence conservation between mouse and human: 0 to ⫺250 bp, ⫺3 to ⫺3.2 kb, and ⫺4.1 to ⫺4.3 kb upstream of the MuRF1 transcription start site (TSS). There were three conserved predicted NF-␬B binding sites located at ⫺3.1, ⫺4.1, and ⫺4.2 kb relative to the mouse MuRF1 TSS. We also found four putative FoxO binding sites at ⫺24 bp, ⫺185 bp, ⫺2.5 kb, and ⫺3 kb relative to the MuRF1 TSS. To determine whether NF-␬B or FoxO binding sites were required for inducing MuRF1 transactivation, a luciferase reporter driven by the 4.4-kb MuRF1 promoter sequence was mutated at all three conserved NF-␬B binding sites (all␬Bmut MuRF1) or at all four conserved FoxO binding sites (allFoxOmut MuRF1). Wild-type and mutated MuRF1 reporter constructs were transiently transfected into rat soleus muscle, and reporter activity was measured 5 days after un-

loading. There was a significant fourfold increase in MuRF1 reporter activity due to unloading, which was abolished in the muscles transfected with the all␬Bmut MuRF1 reporter (Fig. 3), which we previously reported (14). However, mutation of all 4 FoxO binding sites did not abolish reporter activation due to unloading (Fig. 3). We next examined which of the three NF-␬B binding sites are required for inducing MuRF1 expression during unloading atrophy. Each NF-␬B binding site in the 4.4-kb MuRF1 promoter region was mutagenized individually, and then each ␬B site mutant construct was transfected into soleus muscles. Muscles transfected with the MuRF1 reporter mutated at the NF-␬B binding site closest to TSS (␬B3mut MuRF1) showed a twofold upregulation in reporter activity following unloading compared with a fourfold upregulation of the wild-type MuRF1 reporter (Fig. 4). MuRF1 reporter activation due to unloading was abolished in constructs with mutation of either one of the two distal NF-␬B sites (␬B1mut MuRF1 or ␬B2mut MuRF1). DISCUSSION

The key role of MuRF1 in skeletal muscle atrophy has been demonstrated by MuRF1 knockout mice that show attenuated muscle atrophy due to hindlimb unloading (17), denervation (2), and glucocorticoid treatment (1). The MuRF1 gene encodes a muscle-specific E3 ubiquitin ligase that targets, via proteasomal degradation, sarcomeric proteins such as myosin heavy chain (6), myosin binding protein C (MyBP-C), and myosin light chains 1 and 2 (7). Many types of muscle atrophy in vivo are associated with increased MuRF1 expression (1–3, 9, 18, 23, 29) and some consider it a marker of muscle atrophy. However, the transcriptional regulation of the MuRF1 gene is not well understood. Mutagenesis of FoxO and GRE sites in the proximal 500 bp of the MuRF1 promoter showed that these sites were required for MuRF1 activation due to glucocorticoid treatment of Hep2G cells (32). Myogenin knockout significantly attenuated MuRF1 gene upregulation with muscle denervation, and mutagenesis of E-boxes in a proximal MuRF1 promoter (⬍600 bp) suggested that myogenin was required for denervationinduced upregulation of MuRF1 (21). On the other hand, these relatively short MuRF1 promoter fragments were not sufficient for MuRF1 activation due to muscle unloading muscle atrophy (14). Similarly, others showed activation of a 5-kb MuRF1 promoter construct, but not a 500-bp MuRF1 promoter con+1 TSS 100%

Sequence conservation (%)

50% -4 kb

-3 kb

-2 kb

-1 kb

0 kb

NF-κB binding sites FoxO binding sites Fig. 2. Sequence alignment and transcription factor binding sites within the 4.4-kb muscle-specific ring finger protein 1 (MuRF1) promoter region. rVISTA analysis was performed to compare sequence conservation between the mouse and human genomes within the 4.4-kb promoter region of MuRF1. Conservation is show as a histogram; the areas of significant conservation are indicated in pink and the MuRF1 transcription start site (TSS) is indicated by a blue arrow. There were 3 conserved regions between mouse and human sequence located at 0 to ⫺250 bp, ⫺3 kb to ⫺3.2 kb, and ⫺4.1 kb to ⫺4.3 kb upstream of the MuRF1 TSS. In silico analysis of transcription factor binding sites by rVISTA showed 3 NF-␬B binding sites (green boxes), and 4 FoxO sites (blue boxes), which are shown at bottom, plotted against the mouse genome at top. AJP-Cell Physiol • doi:10.1152/ajpcell.00361.2013 • www.ajpcell.org

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XX

kB1 kB2

X

Luciferase

MuRF1

Luciferase

allkBmut MuRF1

* weight bearing hind limb unloading

kB3

XX

XX Luciferase

FBE1 FBE2

allFoxOmut MuRF1

*

FBE3 FBE4

NF-κB binding site

6

4 2 MuRF1 reporter activity (fold change)

0

FoxO binding site

X Site directed mutagenesis

Fig. 3. NF-␬B binding sites, but not FoxO binding sites, were required for MuRF1 promoter reporter activation during muscle unloading. Five days of hindlimb unloading upregulated the wild-type MuRF1 reporter activity by 4-fold. Site directed mutagenesis of all 3 NF-␬B binding sites in the 4.4-kb MuRF1 promoter (␬Bmut MuRF1) abolished MuRF1 reporter activity [data plotted or these 2 mean values were published previously; Open Access Policy PLoS One (14)]. Site-directed mutagenesis of all 4 FoxO binding sites in the 4.4-kb MuRF1 promoter (FoxOmut MuRF1) failed to abolish MuRF1 reporter activity due to unloading. *Statistically different from weight-bearing (P ⬍ 0.05).

struct, in skeletal muscle due to LPS-induced lung injury that leads to atrophy (9). The LPS lung injury also activated an NF-␬B-dependent reporter in muscle as we have demonstrated multiple times in hindlimb muscles of unloaded rats and mice (12, 13, 15, 31). However, primary evidence for the requirement of NF-␬B in the regulation of the MuRF1 gene during atrophy is missing from these studies. Here we provide the first direct evidence that putative NF-␬B sites in the MuRF1 promoter are required for MuRF1 transactivation following unloading-induced muscle atrophy. This is consistent with our previous finding that MuRF1 mRNA upregulation due to unloading is abolished in mice deficient in p50, a transcription factor of the NF-␬B family, or Bcl-3, a p50 transcriptional coactivator (33). Our data suggest that mutation of any one of the ␬B sites can affect MuRF1 expression, particularly the distal two, but all three could be involved in the regulation of the MuRF1 gene with biomechanical unloading. Multiple studies have implicated FoxO transcription factors in the regulation of muscle atrophy in vivo (19, 23, 24). Increased MuRF1 mRNA expression in atrophied muscles due to immobilization, tumor bearing, or sepsis was abolished by

X

Luciferase

MuRF1

Luciferase

kB1mut MuRF1

kB1

X

Luciferase

kB2mut MuRF1

Luciferase

kB3mut MuRF1

kB2

X

kB3

NF-κB binding site FoxO binding site

X Site directed mutagenesis

overexpressing dominant negative FoxO (23, 28), although it is not clear if MuRF1 is a direct or indirect target of FoxO. One study showed that although FoxO is required for MuRF1 expression in limb immobilization, it does not appear to be through DNA binding (27), indicating that MuRF1 may be an indirect target. In the present work, the 4.4-kb MuRF1 promoter construct with mutations in all four putative FoxO binding sites showed that these sites were not required for MuRF1 activation due to muscle unloading. The importance of FoxO transcription factors in disuse atrophy is clear, but MuRF1 appears to be an indirect target of FoxO in unloading atrophy. Although the present data show an essential role for NF-␬B binding sites, but not FoxO binding sites in the regulation of a 4.4-kb MuRF1 promoter construct, it has been shown that direct binding to FoxO sites of the Mafbx1/Fbxo32 promoter is required for its activation (25). We provide definitive genetic data on the relative contributions of the FoxO and NF-␬B cis regulatory elements of the MuRF1 promoter to the expression of this gene in unloading muscle atrophy. However, it is possible that FoxO sites further upstream or downstream from the MuRF1 TSS are involved in regulation of endogenous MuRF1. It is

* weight bearing hind limb unloading

* 0

1

2

3

4

5

MuRF1 reporter activity (fold change)

Fig. 4. Each NF-␬B site abolished or inhibited MuRF1 promoter reporter activity during muscle unloading. Each of the 3 NF-␬B binding sites in the 4.4-kb MuRF1 reporter was mutagenized and then transfected into rat soleus muscles. Mutagenesis of the most proximal NF-␬B site (␬B3mut MuRF1) partially blocked the upregulation of the MuRF1 reporter and mutagenesis of either of the two 5=-distal NF-␬B binding sites (␬B1mut MuRF1 or ␬B2mut MuRF1) abolished activation of the reporter. *Statistically different from weight-bearing (P ⬍ 0.05). AJP-Cell Physiol • doi:10.1152/ajpcell.00361.2013 • www.ajpcell.org

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noteworthy that the discovery of MuRF1 and its role in atrophy was through the use of three models of disuse: immobilization, denervation, and hindlimb unloading (2). Subsequently, other atrophy triggers have been shown to induce MuRF1 upregulation but not in all kinds of muscle atrophy (22, 30). By testing putative regulatory sites via mutagenesis in unloading atrophy, for which several transcription factors have been implicated, we found that NF-␬B sites but not FoxO sites were required for the transcriptional activation of MuRF1, thereby advancing our understanding of the complex physiology of muscle atrophy.

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GRANTS This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-041705 and AR-060217.

16.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). 17.

AUTHOR CONTRIBUTIONS Author contributions: C.-L.W., R.W.J., and E.W.C. performed experiments; C.-L.W. and R.W.J. analyzed data; C.-L.W. and E.W.C. prepared figures; C.-L.W., R.W.J., and S.C.K. approved final version of manuscript; R.W.J. and S.C.K. conception and design of research; R.W.J. and S.C.K. interpreted results of experiments; R.W.J. and S.C.K. edited and revised manuscript; S.C.K. drafted manuscript.

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