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. Author manuscript; available in PMC: 2014 Jun 6.
Published in final edited form as: Cell. 2013 Jun 6;153(6):1366–1378. doi: 10.1016/j.cell.2013.05.015

Regulation of organismal proteostasis by trans-cellular chaperone signaling

Patricija van Oosten-Hawle 1, Robert S Porter 1, Richard I Morimoto 1
PMCID: PMC3955170  NIHMSID: NIHMS479779  PMID: 23746847

Summary

A major challenge for metazoans is to ensure that different tissues each expressing distinctive proteomes are, nevertheless, well protected at an organismal level from proteotoxic stress. We have examined this and show that expression of endogenous metastable protein sensors in muscle cells induces a systemic stress response throughout multiple tissues of C. elegans. Suppression of misfolding in muscle cells can be achieved not only by enhanced expression of HSP90 in muscle cells, but as effective by elevated expression of HSP90 in intestine or neuronal cells. This cell-non-autonomous control of HSP90 expression relies upon transcriptional feedback between somatic tissues that is regulated by the FoxA transcription factor PHA-4. This trans-cellular chaperone signaling response maintains organismal proteostasis when challenged by a local tissue imbalance in folding and provides the basis for a novel form of organismal stress sensing surveillance.

Introduction

The expression of unique combinations of proteins that determine tissue function in metazoans must be maintained by a corresponding tissue-specific network of chaperones and quality control processes to achieve optimal proteostasis in that tissue. For example, the proteostasis network expressed in cells of the immune system, or pancreatic cells that secrete large quantities of proteins is distinct from that expressed in brain or muscle tissues (Powers et al., 2009). This would predict that differences in the proteins expressed in post-mitotic neurons, muscle, or intestinal cells in terms of proteome composition, levels of expression, protein stability, and dynamics, must also have a unique cell-type specific response to extrinsic environmental or physiological stress signals. To counteract such fluctuating conditions, cells employ highly conserved stress responses that monitor the cellular environment and prevent protein mismanagement by restoring proteostasis (Gidalevitz et al., 2011).

Within each cell, this is achieved by the Heat Shock Response (HSR), that upregulates an intrinsic network of molecular chaperones through the activity of HSF-1, a master stress transcriptional regulator (Akerfelt et al., 2010). Activation of the HSR is essential for adaptation and survival at the single cell level. The appearance of multicellularity, however, adds another challenge to maintain proteostasis, as different cell types and tissues need to exchange information to coordinate growth, metabolism, gene expression, and stress responses. For example, in C. elegans the HSR is regulated by thermo-sensory neurons that detect temperature changes to control HSF-1 activity throughout the somatic tissues of the animal (Prahlad et al., 2008). Yet, at the same time, the HSR is associated with numerous tissue-specific human diseases (Mendillo et al., 2012; Morimoto, 2008; Powers et al., 2009). What remains unclear is whether proteotoxic challenges that affect a single cell or tissue, such as the expression of a metastable aggregation-prone or damaged protein, would lead to a strict autonomous response or whether local protein damage within one tissue would be sensed by other tissues as an integrated organismal response.

These questions have led us to ask whether perturbation of proteostasis within a single tissue of C. elegans initiates a response in adjacent tissues. To address this, we used myosin temperature-sensitive (ts) mutations expressed only in muscle and observed induction of the myosin chaperone HSP90 not only in muscle but also in neuronal and intestinal cells. Moreover, cell non-autonomous expression of HSP90 suppressed myosin (ts) misfolding at the restrictive temperature. Consistent with these observations, activation of the HSR in one tissue had beneficial effects in other tissues. These results reveal a compensatory response to a tissue-specific imbalance in proteostasis that functions in a cell non-autonomous fashion in the nematode C. elegans.

Results

Tissue-specific perturbation of proteostasis is recognized at a systemic level

We monitored tissue-specific folding requirements in muscle cells using the HSP90 client protein myosin heavy chain B (UNC-54), an essential component of thick filaments solely expressed in the bodywall muscle of C. elegans (Epstein and Thomson, 1974; Miller et al., 1986). Expression of temperature-sensitive myosin (ts) mutations [unc-54(e1301) or unc-54(e1157)] at the restrictive temperature (25°C) results in misfolded myosin and disruption of thick filaments, leading to severe movement defects and embryonic lethality (Ben-Zvi et al., 2009; Gengyo-Ando and Kagawa, 1991; MacLeod et al., 1977). Because metastable ts proteins are highly dependent on the cellular folding environment (Ben-Zvi et al., 2009; Gidalevitz et al., 2006), we reasoned that expression of unc-54(ts) mutations could place increased demands for chaperones such as HSP90 that are required for folding of myosin and maintenance of muscle function (UNC-54) (Barral et al., 2002; Gaiser et al., 2011) (Figure S1B).

In wild type animals, the sole cytosolic HSP90 (DAF-21) in C. elegans is ubiquitously expressed in the pharynx (ph), intestine (int), pharyngeal nerve ring (n), bodywall muscle (bwm) and the excretory cell (ex), as observed with an hsp90p::GFP transcriptional reporter (Figures 1A and 1B). In unc-54(ts) animals, however, hsp90 mRNA levels are induced almost two-fold at the permissive temperature relative to wild type animals (Figure 1C). Likewise, the hsp90 reporter was induced at the permissive temperature in animals expressing ts alleles of myosin (ts) as well as paramyosin (unc-15, e1402), another component of muscle thick filaments (Miller et al., 1986) (Figure 1I– 1K, Figure S1D–F and S1H–L, respectively) relative to control animals (Figure 1E – 1G and Figure S1C and S1G, J). These results are consistent with increased requirements for HSP90 in bodywall muscle cells (Figure 1K and Figure S1E and S1K). Unexpectedly, the hsp90 reporter was also induced in cells that do not express UNC-54, such as the intestine, pharynx, and excretory cells (Figure 1J–K; Figure S1E–F and S1L).

Figure 1. Tissue-specific perturbation of proteostasis is recognized across multiple tissues in a cell-non-autonomous manner.

Figure 1

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(A,B) Confocal image of a young adult animal expressing the 2.5 kb hsp90 promoter region upstream of GFP (hsp90p::GFP) at 20°C. Pronounced expression was observed in mu ltiple tissues, including pharyngeal muscle (ph), intestine (int), pharyngeal nerve ring (n), bodywall muscle (bwm) and the excretory cell (ex). Scale bar is equal to 100 μm. (B) 63x magnification of the head region. Expression is detected in the bodywall muscle (bwm), the excretory cell (ex), the pharyngeal muscle (ph), pharyngeal nerve-ring (n) and the intestine (int). Scale bar is equal to 10 μm. (C) Total mRNA levels of hsp90 in unc-54(e1301) animals relative to wild type at 15C°C. Bargraphs represent combined mean values of three independent experiments (means ± s.e.m.) **P-value < 0.01. (D–K) hsp90p::GFP reporter expression in unc-54(e1301) animals compared to wild type. Scale bar of figures (D–K) are equal to 100 μm. (F,G) 100x image of wild type expressing the hsp90 reporter in the excretory canal (ex) and bodywall muscle (bwm). (J,K) hsp90 expression in intestinal cells, bodywall muscle and excretory canal in unc-54(e1301) animals. (J) 40x image. (K) 100x image. All fluorescent images were taken at equal exposure times. See also Figure S1.

Thus, these results reveal that disruption of proteostasis by expression of metastable muscle proteins generates a muscle-specific stress that is sensed by multiple tissues in the animal and unexpectedly results in a cell-non-autonomous elevated expression of HSP90.

Tissue-specific increased expression of HSP90 improves the organismal folding environment of myosin (ts) mutants

Since hsp90 expression is induced in muscle cells of myosin (ts) mutants, we asked whether the defective folding of myosin that occurs at the restrictive temperature would be suppressed by increasing the expression of HSP90 in the bodywall muscle. We therefore established C. elegans lines expressing HSP90 (HSP90::GFP) in bodywall muscle cells (HSP90bwm) (Figure 2A). This resulted in an 85% increase of HSP90 above endogenous levels (Figure 2D) that suppressed myosin misfolding and reverted the paralysis of unc-54(ts) mutants at the restrictive temperature (Figure 2E and 2F).

Figure 2. Tissue-specific increased levels of HSP90 improves the organismal folding environment of myosin(ts) mutants.

Figure 2

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(A–C) Confocal images of young adult C. elegans animals overexpressing HSP90::GFP in the (A) bodywall muscle (HSP90bwm), (B) the intestine (HSP90int) or (C) the neurons (HSP90neuro) (scale bar = 100 μm), with 63x magnifications of selected regions (Scale bar = 10 μm). (D) Western Blot analysis of young adult animals overexpressing HSP90 using an anti-C.elegans HSP90 antibody. Levels of HSP90::GFP are normalized to the loading control (tubulin) and relative to endogenous HSP90. (E) Percentage of young adult animals expressing ts myosin [unc-54 (e1301)] alone or in the presence of bodywall muscle-specific HSP90::GFP overexpression (unc-54(ts),HSP90bwm), intestinal overexpression (unc-54(ts),HSP90int), or neuronal overexpression (unc-54(ts),HSP90neuro) showing movement after exposure to restrictive temperature (25°C) for 12 – 24 hours. n = 20 adult animals per strain per experiment. Bargraphs represent the combined results of 3 independent experiments. Error bars = ±SEM. *P-value < 0.05. **P-value < 0.01. (F) Confocal images of the bodywall muscle of age-synchronized wild type, unc-54(e1301), unc-54(e1301),HSP90bwm, unc-54(e1301),HSP90int, and unc-54(ts),HSP90neuro animals after exposure to restrictive temperature (25ºC) for 12 hours, using myo-3::GFP for visualization. Scalebar = 20 μm. See also Figure S2.

Because the hsp90 reporter was also induced in non-muscle tissues, we examined whether increasing the levels of HSP90 in intestinal or neuronal cells would affect the folding of myosin in the muscle cell and confer protection to muscle-specific phenotypes at the restrictive temperature. Therefore, we generated transgenic lines expressing HSP90 in the intestine (HSP90int) or neurons (HSP90neuro) (Figure 2B and 2C). Tissue-specific expression of HSP90::GFP was confirmed by measuring GFP and hsp90 mRNA levels in isolated intestinal cells of HSP90bwm, HSP90int and HSP90neuro (Figure S2F). HSP90::GFP protein levels in the HSP90int and HSP90neuro lines corresponded to an 80% and 45% increase relative to endogenous HSP90, respectively (Figure 2D). Unexpectedly, elevated expression of HSP90 in the intestine or the neurons also suppressed muscle fiber degeneration at restrictive temperature (Figure 2F), improved the motility of unc-54(ts) mutants (Figure 2E), and alleviated embryonic lethality (Figure S2E).

Thus, the observation of induced hsp90 expression in non-muscle tissues of myosin (ts) or paramyosin (ts) mutants (Figure 1D–K and Figure S1C–L), indeed serves as a protective physiological response that improves the folding environment of challenged muscle cells and enhances organismal viability of myosin (ts) animals during chronic proteotoxic stress.

Tissue-specific expression of HSP90 blocks the HSR in distal tissues

The ability of elevated levels of HSP90 to establish a protective folding environment for muscle cells in a cell non-autonomous manner in myosin (ts) mutants, led us to consider whether transgenic HSP90 overexpression lines were also cross-protected against more severe heat stress conditions. In wild type animals, a stringent heat shock regimen at 35°C for 10 hours results in 20% survival, whereas by comparison all three transgenic HSP90 lines were extremely hypersensitive to heat stress with less than 5% survival at the 10 hour time point (Figure 3A). This corresponds to the same level of heat stress sensitivity exhibited by hsf-1(sy441) hypomorph mutant animals (Figure 3A) (Hajdu-Cronin et al., 2004). Thus, elevated levels of HSP90, while protective under chronic ambient proteotoxic stress due to the expression of metastable proteins, was not tolerated under severe acute stress conditions. This suggests that metazoan cells employ a novel form of trans-cellular communication to maintain tissue proteostasis that is protective during mild fluctuating environmental conditions, but is deleterious when animals are challenged by a severe heat shock.

Figure 3. Elevated tissue-specific HSP90 levels repress the HSR at an organismal level.

Figure 3

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(A) Thermosensitivity of young adult animals (n = 100) with indicated genotypes exposed to 35°C heat stress. *P-value < 0.05. **P-value < 0.01. (B) Total mRNA levels of hsp70 (C12C8.1 and F44E5.4) and hsp16 (hsp-16.2) after heat shock (1h at 33°C) in young adult myo-2p::mCherry, hsf-1 (sy441) mutant, HSP90neuro, HSP90int, HSP90bwm animals and upon RNAi-mediated GFP or hsp90 knockdown prior to heat shock in the transgenic HSP90 lines, relative to wild type. (A and B) Bargraphs represent combined mean values of three independent experiments (means ± s.e.m.) (C)

DIC Nomarski images of (i) wild type, (v) HSP90bwm, (ix) HSP90int and (xiii) HSP90neuro animals expressing the hsp70p::mCherry reporter. Expression of the hsp70p::mCherry reporter 7 hours after heat shock (33°C, 1h) in representative (ii) wild type and in (vi) HSP90bwm, (x) HSP90int and (xiv) HSP90neuro animals. Yellow arrow in vi, x, and xiv indicates the pharyngeal myo-2::mCherry co-injectionmarker, present in all transgenic HSP90::GFP lines. (iii,vii,xi,xv) 20x magnifications of the posterior region of (iii) wild type and HSP90 overexpression lines (vii, xi, xv), indicating hsp70 induction in spermatheca (sp), bodywall muscle (m) and the intestine (i). (iv, viii, xii, xvi) HSP90::GFP expression in true color. Yellow arrows in (xvi) indicate neuronal cells expressing HSP90::GFP. (i–xvi) Scale = 100 μm. See also Figure S3.

One explanation for the stress hypersensitivity in animals overexpressing HSP90 in specific tissues could be that higher levels of HSP90 has inhibitory effects on the induction of the HSR. To address this, we quantified the expression of three representative HS genes corresponding to two heat-inducible hsp70s (C12C8.1 and F44E5.4) and the small heat shock protein hsp16 (hsp-16.2). Relative to wild type animals, the HSR in HSP90bwm animals was suppressed 20-fold, and in HSP90int and HSP90neuro animals suppressed five- and three-fold, respectively (Figure 3B). This inhibition of the HSR, by tissue-specific expression of HSP90, was equivalent to that observed for the hsf-1 (sy441) hypomorph mutant (Figure 3B). Moreover, the HSR was fully restored in these transgenic lines by reducing the levels of HSP90::GFP using GFP RNAi or hsp90 RNAi (Figure 3B). The inability to mount an organismal HSR was also not due to increased expression of other chaperones that could negatively regulate HSF-1 (Morimoto, 1998), as basal levels of constitutive hsp70 (hsp-1), inducible hsp70 (C12C8.1), and hsp16 were comparable in wild type and HSP90 overexpression lines (Figure 6A).

Figure 6. A PHA-4 – dependent transcriptional feedback coordinates cell-non-autonomous hsp90 expression.

Figure 6

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(A) Total mRNA levels of hsp-1 (constitutive hsp70), hs-inducible hsp70 (C12C8.1 and F44E5.4), small heat shock protein hsp16 (hsp-16.2) and hsp90 in HSP90 overexpression lines at 20°C relative to wild type. The slightly increased hsp levels in the HSP90bwm may be indicative of the higher sensitivity of muscle cells to proteostatic perturbation, in line with the observations on tissue-specific hsp90 knockdown, where the HSR is primarily induced in the bodywall muscle. (B) Organismal hsp90 expression in HSP90 overexpression lines is independent of hsf-1. hsp90 mRNA levels in control (EV) and animals fed with hsf-1 RNAi. Whereas hsp90 expression in wild type is hsf-1 dependent, RNAi-mediated knockdown of hsf-1 leaves organismal hsp90 levels in HSP90bwm, HSP90int or HSP90neuro unchanged. (C) pha-4 RNAi decreases elevated hsp90 expression in the overexpression lines. (D) hsp90p::GFP reporter expression in myosin (ts, e1157) or paramyosin (ts, e1402) mutants is reduced during pha-4 RNAi when compared to control RNAi (EV). Scalebar = 50 μm. (E) pha-4 activity is increased in the HSP90 overexpression lines. mRNA levels of pha-4 regulated genes sod-1, sod-2, sod-4 and sod-5 are induced in eat-2(ad1113) mutants, HSP90bwm, HSP90int and HSP90neuro animals relative to wild type. *P-value < 0.05. **P-value < 0.02. (F) pha-4 mRNA expression levels in eat-2 and HSP90 overexpression lines are upreguated, relative to the wild type control. *P-value < 0.05. (G) pha-4 activity in intestinal cells of eat-2(ad1113), HSP90bwm, HSP90int and HSP90neuro animals relative to wild type. **P-value < 0.02. (H) pha-4 mRNA expression levels are induced in the intestines of eat-2 mutants and HSP90int (=signaling tissue), but not in the intestines of HSP90bwm or HSP90neuro (=recipient tissue). *P-value < 0.05. (n.s. = not significant). (A – H) All bar graphs represent combined mean values of three independent experiments (3 biological replicates) (means ± s.e.m.). See also Figure S6.

To identify the molecular step at which HSP90 inhibits the organismal induction of the HSR, we examined the regulation of HSF-1 DNA binding activity by electrophoretic gel mobility shift assays in heat shocked wild type, HSP90bwm and HSP90neuro animals. The level of HSF-1 DNA binding activity in heat shocked wild type extracts was strongly induced relative to HSP90bwm and HSP90neuro animals that showed an at least two-fold reduction of HSF-1 DNA binding (Figure S3A and S3B; see Extended Experimental Procedures). This was not due to any detectable changes in the expression of hsf-1 mRNA levels relative to wild type animals (Figure S3C). Because HSP90 functions as a negative regulator of HSF-1 (Zou et al., 1998), we conclude that local changes in the levels of HSP90 induces a cell non-autonomous regulatory process that inhibits HSF-1 activation in distant tissues. Thus, the molecular consequence of locally elevated HSP90 expression is a systemic reduction in HSF-1 DNA binding activity, leading to a global reduction of the HSR.

Local changes in HSP90 levels inhibit HSP expression in distal tissues

In order to directly monitor the effects of local HSP90 overexpression on the organismal HSR in living animals, we employed an hsp70p::mCherry (C12C8.1p::mCherry) reporter strain to visualize the HSR across the different tissues. Heat shock induction of hsp70p::mCherry was readily detected in the spermatheca (sp), the bodywall muscle (m) and the intestine (i) (Figure 3C, i–iv) of wild type animals. In contrast, animals expressing HSP90::GFP in bodywall muscle (Figure 3C, v–viii and Figure S3D, i), intestine (Figure 3C, ix–xii and Figure S3D, iv) and neurons (Figure 3C, xiii–xvi and Figure S3D, vii) showed a reduction in HS-inducibility of the hsp70p::mCherry reporter in multiple tissues. For example, increased expression of HSP90 in muscle cells (Figure 3C, viii, green) blocked induction of the hsp70 reporter not only in muscle cells but also in intestinal cells (Figure 3C, vii) relative to wild type animals (Figure 3C, iii). Likewise, in HSP90int animals, the hsp70 reporter was not induced in the intestine and induced only slightly in muscle cells (Figure 3C, xi). Consistent with the other transgenic HSP90 lines, animals overexpressing neuronal HSP90 also showed very little induction of the hsp70 reporter in both spermatheca and muscle, and no induction of the HSR in intestinal cells (Figure 3C, xv and Figure S3D, vii).

The compromised HSR could be restored to wild type levels in the individual tissues by reducing overall HSP90 levels by hsp90 RNAi (S3D, ii, v, viii) or HSP90::GFP levels by GFP RNAi (S3D, iii, vi, ix) prior to HS (Figure S3D, i–ix). The three- and five-fold decreased expression of the hsp70p::mCherry reporter in HSP90bwm and HSP90int animals respectively, was rescued by hsp90 or GFP RNAi (Figure S3E). The observation that hsp90 or GFP RNAi only minimally restored hsp70 reporter expression in HSP90neuro, is consistent with the measurement of mRNA levels of HS induced genes (Figure S3D, viii, ix and S3E, and Figure 3B, respectively), and that neurons are less susceptible to RNAi (Simmer et al., 2002).

To further characterize the HSR in tissues that were not targeted for overexpression of HSP90, and to rule out that the hsp70p::mCherry transgene interfered with endogenous HS gene expression, we isolated intestinal tissue from heat-shocked HSP90bwm, HSP90int and HSP90neuro animals that lack the hsp70p::mCherry reporter transgene and quantified the expression of endogenous hsp70 mRNA relative to intestinal cells from wild type animals. The inducible expression of hsp70 was reduced two-fold in isolated intestinal cells of HSP90bwm, 20-fold in the intestine of HSP90int and five-fold in HSP90neuro, relative to wild type levels (Figure S3F). These results are consistent with the reduction of hsp70p::mCherry fluorescence in the intestines of respective strains (Figure 3C, iii compared to vii, xi and xv, respectively) and provide supportive evidence that the localized expression of HSP90 has global inhibitory effects on the organismal HSR.

Taken together, these results show that increased levels of HSP90 in a single tissue has cell-non-autonomous inhibitory effects on HSP expression in other tissues within the organism, and that tissue-specific perturbations of the proteostasis network have consequences throughout the organism.

Our results reveal a potential conundrum: whereas elevated HSP90 levels in non-muscle tissues can cell-non-autonomously rescue the muscle-specific phenotype of myosin (ts) mutants, tissue-specific elevated levels of HSP90 are detrimental under severe heat stress conditions through cell-non-autonomous repression of HSF-1 transcriptional activity. One explanation for repression of HSF-1 activity in non-target tissues, or improved myosin folding when HSP90 is expressed in non-muscle tissues, is that HSP90 overexpressed in one tissue is released and taken up by surrounding cells where they could interact with client proteins such as myosin or HSF-1. In C. elegans, proteins secreted from a cell enter the pseudocoelomic space, a bodycavity exposed to all tissues of the animal, before they can be taken up by surrounding tissues (Altun-Gultekin, 2009). Thus, materials secreted in the pseudocoelom are taken up non-specifically by coelomocytes, scavenger cells that perform a primitive surveillance function in the animal (Altun-Gultekin, 2009; Fares and Greenwald, 2001). Therefore, we examined whether HSP90::mCherry overexpressed in neurons, bodywall muscle or intestine are secreted into the pseudocoelomic space and subsequently endocytosed by coelomocytes using a strain expressing GFP::RAB-5 under the control of a coelomocyte promoter (Sato et al., 2005), to image an uptake of HSP90::mCherry into coelomocytes. However, HSP90::mCherry fluorescence was not detected in coelomocytes, suggesting that overexpressed HSP90 is not exported into the extracellular space (Figure S3G). Thus rather than intercellular transmission of HSP90, the cell-non-autonomous effect giving rise to improved myosin maintenance or repression of HSF1 in non-target tissues must be achieved by a different mechanism.

Tissue-specific knockdown of HSP90 induces a systemic organismal HSR

Having demonstrated that increased expression of HSP90 in any single tissue leads to the repression of HSF-1 activity throughout the animal, we reasoned that tissue-specific hsp90 RNAi should result in induction of the HSR in multiple tissues. To accomplish tissue-specific knockdown of hsp90, we employed the sid-1 mutation (Winston et al., 2002), which allows cell-autonomous RNAi but is defective for systemic RNAi (Winston et al., 2002). To confirm that HSP90 levels were reduced in specific tissues, animals expressing the hairpin construct in muscle (hp-hsp90bwm), intestine (hp-hsp90int) or neurons (hp-hsp90neuro) were crossed with HSP90::mCherry lines (Figure S4A–F). Quantitation of mCherry fluorescence intensity shows that HSP90 levels are decreased significantly only in the targeted tissue, albeit with slight variation among animals (Figure S4A–F). For example, muscle-specific knockdown of hsp90 (hp-hsp90bwm) reduced HSP90::mCherry fluorescence in HSP90bwm animals to 55%, relative to control animals, whereas HSP90::mCherry expression in HSP90int and HSP90neuro animals were unaffected by muscle-specific hairpin RNAi (Figure S4A and S4B). Likewise, hsp90 hairpin RNAi expressed in the intestine (hp-hsp90int) or the neurons (hp-hsp90neuro) reduced HSP90 levels to 25% in only HSP90int and to 50% in HSP90neuro animals, respectively (Figure S4C–D and S4E–F, respectively).

Tissue-specific knockdown of hsp90 in the neurons, intestine or bodywall muscle also resulted in significant developmental delays (Figure S4G), and the appearance of diverse aberrant phenotypes (Figure S4H) consistent with the proposed roles of HSP90 in development, signal transduction, gene expression (Taipale et al., 2010), and as a capacitor of phenotypic variation (Queitsch et al., 2002; Rutherford and Lindquist, 1998).

Consistent with the function of HSP90 as a repressor of the HSR, RNAi-mediated knockdown of hsp90 in a single tissue induced the expression of hsp70, under normal conditions of growth (Figure 4A), corresponding to a 10-fold induction of hsp70 mRNA (C12C8.1 and F44E5.4) in hp-hsp90bwm animals and 8-fold up-regulation in the hp-hsp90int and hp-hsp90neuro lines. By comparison, knocking down hsp90 in all tissues of wild type animals by systemic RNAi resulted in a ~30 and 15-fold induction, respectively of two hsp70 genes (C12C8.1 and F44E5.4) (Figure 4A). This induction of hsp70 in the tissue-specific hsp90 knockdown lines was sufficient to ameliorate organismal survival compared to the control line (Figure 4B), indicating that the induction of hsp70 in multiple tissues was protective.

Figure 4. Tissue-specific knockdown of hsp90 cell-non-autonomously induces the HSR.

Figure 4

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(A) Bodywall-muscle, intestine- and neuron-specific hsp90 RNAi induces basal levels of hsp70 (C12C8.1 and F44E5.4) expression at 20°C, compared to control animals (sid-1). Wild type animals allow import of dsRNA from surrounding tissues, leading to higher induction of organismal hsp70 than in the tissue-specific knockdown lines. Bargraphs represent combined mean values of three independent experiments (means ± s.e.m.) **P-value < 0.05. (B) Thermosensitivity of young adult animals (n = 100) expressing the indicated tissue-specific hp-hsp90 construct exposed to 35 °C heat stress. Bargraphs represent combined mean values of three independent experiments (means ± s.e.m.) **P-value < 0.01. *P-value < 0.05. (C) Tissue-specific knockdown of hsp90 induces expression of the hsp70 reporter (hsp70p::mCherry) at 20°C. DIC images of synchronized young adult (i) control animals (sid-1), (v) hp-hsp90bwm, (ix) hp-hsp90int, and (xiii) hp-hsp90neuro animals expressing the hsp70 reporter. Expression of the hsp70p::mCherry in (ii) sid-1 control animals, (vi) hp-hsp90bwm, (x) hp-hsp90int, and (xiv) hp-hsp90neuro. (iii, vii, xi, xv) 20x magnification of control (iii) and tissue-specific hsp90 knockdown lines (vii, xi, xv) indicating expression of hsp70p::mCherry in the pharynx (ph), intestine (int) and bodywall muscle (m). (iv, viii, xii, xvi) Overlay of DIC Nomarski and hsp70p::mCherry (red). Scalebars = 100 μm. See also Figure S4.

We examined the induction of the HSR at the level of individual tissues by monitoring the hsp70p::mCherry reporter in living animals expressing the tissue-specific hsp90 knockdown constructs, and observed that the HSR was induced not only in the primary tissue but also in distal tissues, that were not targeted by the hairpin RNAi (Figure 4C). Knockdown of hsp90 in the bodywall muscle significantly up-regulated hsp70 expression not only in muscle cells, but also in the intestine and pharynx (Figure 4C, vi, vii and viii, red). Likewise, in animals with reduced levels of HSP90 in the intestine, we observed elevated hsp70 expression in the intestine and muscle cells (Figure 4C, x, xi and xii, red). Animals expressing hairpin hsp90 dsRNA in neurons however exhibited an increased hsp70 expression in only bodywall muscle cells (Figure 4C, xiv, xv and xvi, red). These results are consistent with the observation that systemic knockdown of hsp90 in wild type animals induces the HSR primarily in muscle tissue, such as the bodywall muscle, pharynx and the vulval muscle (Figure S4I), corroborating previous observations that bodywall muscle cells may be more sensitive to a reduction of hsp90 than other tissues (Gaiser et al., 2011).

In conclusion, either enhancing or suppressing the levels of HSP90 within a single tissue has complementary effects on the induction of the HSR across adjacent tissues of C. elegans. This indicates the involvement of a cell-non-autonomous regulatory mechanism that modifies organismal HSF-1 activity in response to tissue-specific alteration of HSP90 levels.

HSP90 expression is regulated in a cell-non-autonomous manner, independent of neuronal activity

The cell-non-autonomous effect of HSP90 on myosin maturation and organismal HSF-1 activity poses an interesting question of how HSP90 is regulated in C. elegans. Expression of the hsp90 reporter (hsp90p::GFP) was up-regulated across multiple tissues when HSP90 levels were elevated in a single tissue. As shown in Figure 5A, increased expression of HSP90 in bodywall muscle (Figure 5A, v–viii,) or intestine (Figure 5A, ix – xii) resulted in induction of the hsp90 reporter in pharynx, excretory cell and intestine (Figure 5A, vi – vii and x – xi, respectively). Likewise, elevated HSP90 levels in the neurons increased endogenous hsp90 expression in the intestine, pharynx and bodywall muscle (Figure 5A, xiv – xv). Thus the increased activity of the transcriptional hsp90 promoter::GFP fusion indicates the involvement of a transcriptional regulatory mechanism that cell-non-autonomously regulates endogenous hsp90 expression in response to a tissue-specific imbalance.

Figure 5. Coordination of cell-non-autonomous hsp90 expression is regulated independent of neural control.

Figure 5

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(A) Tissue-selective increased levels of HSP90 up-regulates expression of the transcriptional hsp90p::GFP reporter at normal conditions (20°C). DIC images o f (i) wildtype, (v) HSP90bwm, (ix) HSP90int and (xiii) HSP90neuro expressing the hsp90p::GFP reporter. hsp90 reporter expression in representative whole animals (ii, vi, x, xiv) and magnified head region (iii, vii, xi, xv) in (ii, iii) wild type, (vi, vii) HSP90bwm, (x, xi) HSP90int, and (xiv, xv) HSP90neuro, indicating hsp90p::GFP in the pharynx (ph), the intestine (int), bodywall muscle (m) and excretory cell (ex). (iv, viii, xii, xvi) HSP90::mCherry (red) expression in true color. Yellow arrows in (iii, vii, xi and xv) indicate hsp90p::GFP expression in the excretory cell. Scalebars = 100 μm.(B) Schematic representation of the major modes of neuro-secretion in C. elegans, regulated via either dense core vesicles (DCV) through unc-31 or via small core vesicles (SCV) through unc-13. (C) Total hsp90 mRNA levels of HSP90 overexpression lines in an unc-31 deletion mutant background (white bars) or crossed to an unc-13 deletion mutant (light grey bars) relative to control animals (dark grey bars). Bargraphs represent combined mean values of three independent experiments (means ± s.e.m.). See also Figure S5.

Since neurons are important for information exchange and coordination of transcriptional regulation at the organismal level (Prahlad et al., 2008), we examined whether neuronal signaling was essential for the cell-non-autonomous regulation of hsp90 expression. We therefore tested whether inhibition of the major modes of neuro-secretion, the dense core vesicle (DCV) release of neurotransmitter and the small core vesicle (SCV) release of neuropeptides (Richmond and Broadie, 2002), suppressed the transcriptional tissue-feedback in response to elevated tissue-specific HSP90 (Figure 5B), since DCV-dependent neurosecretion is also required to maintain optimal levels of chaperones in non-neuronal tissues (Prahlad and Morimoto, 2011). Organismal levels of hsp90 mRNA were unchanged through inhibition of SCV via deletion of unc-13 (Kohn et al., 2000), as well as through inhibition of DCV via deletion of unc-31 (Hammarlund et al., 2008; Speese et al., 2007) (Figure 5C), which correlated with hsp90p::GFP expression throughout tissues (Figure S5). These results indicate that cell-non-autonomous regulation of hsp90 expression is independent of neuronal signaling, and therefore communicated directly between somatic tissues.

PHA-4 dependent transcriptional response regulates cell-non-autonomous hsp90 expression

To examine the cell-non-autonomous regulation of hsp90, we addressed the role of HSF-1, the major stress-inducible transcription factor (Akerfelt et al., 2010). hsp90 expression in wild type animals was reduced upon hsf-1 RNAi (Figure 6B). However, consistent with repression of HSF-1 transcriptional activity in the HSP90 overexpression lines (Figure 3 and Figure 6A), treatment with hsf-1 RNAi did not affect the levels of hsp90 in HSP90bwm, HSP90int or HSP90neuro lines (Figure 6B), revealing an HSF-1 independent process.

We next turned our attention to data from the modENCODE project that identified DAF-16, SKN-1, DAF-12 and PHA-4 binding to the hsp90 promoter by ChIP-Seq ((Celniker et al., 2009); (http://modencode.oicr.on.ca/fgb2/gbrowse/worm/)). Many of these factors also have established roles in proteostasis (Hsu et al., 2003; Morley and Morimoto, 2004; Oliveira et al., 2009; Panowski et al., 2007; Wang et al., 2010; Zhong et al., 2010). Of these, RNAi-mediated knockdown experiments identified pha-4 to have the strongest reduction of organismal hsp90 expression in both wild type and all three HSP90 overexpression lines (Figure 6C). pha-4 RNAi also correlated with reduced expression of the hsp90 reporter across multiple tissues in the HSP90 overexpression lines (Figure S6A). These results suggest that PHA-4 is necessary for increased cell-non-autonomous hsp90 expression. Moreover, pha-4 RNAi suppressed the induction of hsp90 in myosin (ts) and paramyosin (ts) mutants back to lower wild type levels (Figure 6D).

Thus, a tissue-specific imbalance through increased levels of HSP90 or the expression of a metastable client leads to a PHA-4 dependent transcriptional feedback between different tissues that coordinates and balances expression of HSP90 throughout the animal. This cell-non-autonomous transcriptional response regulated by PHA-4 is beneficial during mild chronic proteotoxic stress as in the case of myosin (ts) mutants that require higher levels of HSP90, but can become detrimental under severe HS conditions as up-regulated hsp90 expression through this transcriptional mechanism also represses the HSR (Figure S6B and S6C). Consistent with this result, a tissue-specific imbalance through reduced hsp90 expression, that induces the HSR in different tissues (Figure 4C) also requires functional PHA-4 for this inter-tissue response, as demonstrated by using a pha-4(zu225);smg-1 mutant (Gaudet and Mango, 2002) (Figures S6D and S6E).

To further investigate the role of PHA-4 in this inter-tissue communication, we examined pha-4 activity and expression levels in the HSP90 overexpression lines. Pha4 activity was measured by examining the levels of the pha-4 regulated sod genes (sod-1, sod-2, sod-4, sod-5) that contain a PHA-4 consensus binding site in the respective promoters (Panowski et al., 2007) as confirmed by modENCODE. All three HSP90 overexpression lines exhibit increased pha-4 activity (Figure 6E) as well as elevated levels of pha-4 mRNA (Figure 6F), comparable to the long-lived eat-2 mutant that harbors intrinsically higher pha-4 activity and mRNA levels relative to wild type animals (Panowski et al., 2007). Thus, the higher activity and expression levels of pha-4 in response to a tissue-specific imbalance is consistent with the observation that functional PHA-4 is required for the systemic effects through trans-cellular chaperone signaling.

To understand how this inter-tissue signaling is regulated in the receiving tissue, we examined pha-4 activity and expression levels in isolated intestinal cells of the HSP90 overexpression lines (Figure 6G and 6H). Whereas pha-4 activity (Figure 6G) and expression levels (Figure 6H) are induced in the intestinal cells of HSP90int animals (i.e. signaling tissue), pha-4 activity but not mRNA levels are increased in the receiving tissue (i.e. intestines of either HSP90neuro or HSP90bwm) (Figures 6G and 6H). This reveals that pha-4 expression and activity are required in the signaling tissue and suggests two possibilities for the requirement of PHA-4 in the receiving tissue: that PHA-4 has higher activity despite being expressed in relatively lower amounts, or that PHA-4 in the signaling tissue activates a downstream signaling cascade that acts independently of PHA-4 in the receiving tissue to regulate gene expression (see also Figure 7). Thus, PHA-4 or pha-4 dependent downstream signaling likely adopts a more general role in trans-cellular chaperone signaling as a regulatory effector that contributes to organismal proteostasis surveillance.

Figure 7. Model for the cell non-autonomous regulation of HSP90 expression in C. elegans.

Figure 7

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An imbalance of the proteostasis network through the presence of metastable myosin increases the expression of HSP90 in muscle cells but also in different cell-types, such as the intestine. This is regulated by PHA-4 and communicated through trans-cellular chaperone signaling to other tissues, independent of neural activity. Increased pha-4 activity is required in the signaling and receiving tissue, however pha-4 may act from a distance to regulate gene expression in the receiving tissue via a down-stream signaling cascade. The resulting highly abundant HSP90 levels in the entire animal are beneficial for myosin folding in unc-54(ts) mutants, but can become detrimental during severe heat shock, due to cell non-autonomous repression of HSF-1 transcriptional activity. Likewise, a tissue-specific perturbation through reduced hsp90 levels (lower panel) leads to induction of the HSR in the same and recipient tissues. Transduction of the response to the recipient tissue is also pha-4 dependent.

Discussion

Local perturbations of the proteostasis network, whether caused by tissue-specific expression of metastable proteins, or by the elevated expression of individual chaperones such as HSP90, are compensated by a beneficial trans-cellular chaperone signaling response from adjacent tissues in C. elegans. This suggests that the unique complement of proteins expressed in each tissue is maintained by a combination of autonomous and non-autonomous quality control processes to prevent misfolding and aggregation from dominating the health of a tissue. We propose that individual tissues within an organism serve not only as sensors that respond to disruption of their own cell-specific proteostasis networks, but also to function as sentinels to disseminate local proteotoxic challenges to tissues within the organism to mount a protective response.

A model to describe how such compensatory responses in different tissues can protect the organism from environmental fluctuations to ensure survival of animals harboring genetic pre-dispositions for protein misfolding is shown in Figure 7. Disturbance of the tissue-specific proteostasis network by expression of a metastable client protein such as temperature-sensitive myosin induced the expression of HSP90 not only in muscle tissue, but also in distal tissues. This response is regulated by PHA-4 activity and communicated to other tissues by trans-cellular chaperone signaling. Indeed, increased expression of HSP90 at the organismal level is beneficial for the folding of myosin (ts) mutants under mild temperature stress (Figure S6B and Figure 7). However, HSP90 also represses HSF-1 transcriptional activity (Bharadwaj et al., 1999; Zhao et al., 2002); therefore, elevated levels of HSP90 result in a failure to mount a HSR in multiple tissues upon exposure to severe heat shock, thus affecting organismal survival (Figure S6C and Figure 7). Our results also show that increased levels of HSP90, by inhibiting HSF-1, override the neuronal signal that regulates the HSR. When HSP90 levels are elevated in a specific tissue, cell-non-autonomous regulation of endogenous HSP90 expression is uncoupled from neural regulation of HSF-1 activity and is henceforth regulated by the FoxA transcription factor PHA-4. Although the molecular nature of the intercellular signal that mediates this trans-cellular signaling response between tissues is unclear, it is dependent upon PHA-4 (Figures 6 and S6), revealing a more general role for this transcription factor as a regulatory effector. Moreover, pha-4 expression and activity are increased in the signaling tissue harboring higher hsp90 levels, which leads to activation of gene expression in the downstream recipient tissues. We speculate that regulation of hsp90 expression by PHA-4 under the conditions reported by modENCODE and the inter-tissue response, functions at low basal levels in wild type animals and becomes activated in response to tissue-specific perturbations to restore organismal proteostasis.

Trans-cellular chaperone signaling therefore communicates a local proteotoxic stress event to adjacent cells and tissues, thus providing a community-level response. By this, we propose that metazoans have developed a survival strategy to prevent the “weakest link” from compromising organismal health and survival. This form of regulation in which the community of adjacent cells and tissues restores proteostasis is distinct from the neuronal control of the HSR that transmits an external environmental signal through the thermo-sensory AFD neuron to coordinate the regulation of HSF-1 activity in non-neuronal tissue (Prahlad et al., 2008). Yet, the two forms of cell non-autonomous regulation complement to provide a protective mechanism for the unique proteomes expressed in different cells and tissues. Such modulation of proteostasis between non-neuronal tissues in C. elegans could similarly be achieved via exchange of small signaling molecules such as metabolites, ROS, peptides or small regulatory RNA molecules (Belting and Wittrup, 2008) that activate and change tissue-specific transcriptional programs in the target tissues.

Among the molecular chaperones, fluctuations in HSP90 levels have been shown to affect developmental robustness in Drosophila and Arabidopsis (Gangaraju et al., 2011; Queitsch et al., 2002; Rutherford and Lindquist, 1998). Systemic reduction of HSP90 results in larval arrest and in greater penetrance of mutations in C. elegans (Burga and Lehner, 2012; Casanueva et al., 2012), whereas gain of function mutations cause defective dauer signaling (Birnby et al., 2000), and defects in muscle cells (Gaiser et al., 2011). Our results provide additional support that tissue-specific reduction of HSP90 leads to cell-non-autonomous developmental defects and phenotypes that have not been previously associated with HSP90 dysfunction. For example, RNAi-mediated knockdown of hsp90 in muscle cells exposes defects in other tissues, such as the excretory canal (exc) phenotype, aberrant hermaphrodite tail formation, or abnormal intestines (Figure S4H). These observations lend support for a role of HSP90 to integrate transcriptional response across different cell types and tissues in C. elegans, consistent with a role in a complex systems network at the hub of diverse signaling processes from yeast to mammals (Taipale et al., 2010).

While our studies have only addressed the role of HSP90 with regard to folding and stability of the client protein myosin, and how altered levels of HSP90 transmits a signal across cells; likewise, other chaperones may also have similar effects on organismal responses. In particular, client-specific responses could ensure that different molecular chaperones could regulate complementary types of proteotoxic stress signaling events.

In summary, the work presented here provides the basis of a new mechanism of how tissues within an organism respond to disturbances of proteostasis to regulate a cell-non-autonomous control of chaperone expression that restore balance between tissues. Future studies will address how tissue-specific perturbations in a limited number of sensor cells are transmitted to the recipient cells and tissues and whether trans-cellular chaperone signaling observed in C. elegans extends to other metazoans.

Experimental Procedures

Heat shock

Synchronized populations of C. elegans strains were grown at 20°C and animals were heat shocked at a population density of 10–15 young adult animals per plate as described (Prahlad et al., 2008). Animals were heat shocked by sealing plates with parafilm and zip-lock bags and immersing into a water bath equilibrated at 33°C for 1 hour or at 34°C for 30 minutes and allowed to recover for 1 hour at 20 °C before they were harvested for quantitative RT PCR. Each qRT-PCR experiment was repeated in triplicate.

Thermotolerance

For thermotolerance assays, a synchronized population of approximately 20 young adult animals on each plate was placed into a 35°C incubator (Fisher Scientific – Isotemp Incubator). 5 samples, each consisting of 20 adult animals were used for one time-point and the experiment was repeated at least 3 times (3 biological replicates) to achieve substantial N values. Statistically significant changes in survival were considered when p<0.05 (Student’s T-test). Plates were collected at the indicated time-points (8 hours and 10 hours) and animals were allowed to recover for 2 hours at 20 °C before scoring for touch-induced movement and pharyngeal pumping.

Assays for temperature-sensitive phenotypes

For the paralysis assay of unc-54(ts) mutants, 20 young adult animals were placed onto fresh NGM plates at 25°C and scored 12 hours later for touchinduced movement. For the survival assays of unc-54(ts) mutants, young adult animals were allowed to lay eggs at 25°C for 3 hour s. After removal of the adults, plates were incubated at the restrictive temperature for 24 – 48 hours and then scored for surviving and moving progeny (n = 50). All experiments were repeated three times (3 biological replicates). Statistically significant changes in movement were considered if p<0.05 (Student’s T-test).

RNAi experiments

For RNAi-mediated knockdown of indicated genes, synchronized populations of nematodes were placed on E. coli strain HT115(DE3) transformed with appropriate RNAi vectors (J. Ahringer, University of Cambridge, Cambridge, U.K.) as decribed previously (Nollen et al., 2004).

To knock-down hsp90 prior to heat shock, 30 L4 larvae were placed on E. coli strain HT115(DE3) transformed with hsp90 RNAi.

Supplementary Material

01
02

Highlights.

  • Imbalance of muscle proteostasis induces chaperone expression in different tissues

  • Tissue-specific modulation of chaperones such as HSP90 affects the organismal HSR

  • This intertissue response is regulated by interplay between HSF-1 and PHA-4

  • Trans-cellular chaperone signaling integrates cell-specific and organismal responses

Acknowledgments

We thank the Caenorhabditis Genetics Center (University of Minnesota) for strains used in this study, the BC C. elegans Gene Expression Consortium for strain BC10293, Dr. Daniel Czyz for the hsp70pr::mCherry reporter strain (AM722) and Dr. P.W. Piper and Dr. S. Millson (University of Sheffield, UK) for providing yeast strains and plasmids. These studies were supported by an Erwin-Schroedinger postdoctoral fellowship (Austrian Science Fund) and from the Chicago Center for Systems Biology Postdoctoral Fellowship supported by NIGMS P50 grant (P50-GM081192) and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community trust to PVOH, and grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, the Chicago Biomedical Consortium and the Daniel F. and Ada L. Rice Foundation to RIM.

Footnotes

Supplemental Information

Supplemental Information includes Extended Experimental Procedures and six figures.

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