© 2014. Published by The Company of Biologists Ltd.
Kukic et al., Page 1 of 33
1 2 3 4 5 6 7
Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity.
8
Journal of Cell Science
Accepted manuscript
9
Ira Kukic1, Shannon L. Kelleher2,3,4 and Kirill Kiselyov1
10 11 12
1
13
15260 USA,
14
Development, The Pennsylvania State University, University Park, Pennsylvania 16802, USA,
15
Department of Surgery, Penn State Hershey Medical Center, Hershey, Pennsylvania 17033,
16
USA, and
17
Center, Hershey, Pennsylvania 17033, USA.
From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania
4
2
the Department if Nutritional Sciences, College of Health and Human 3
Department of Cellular and Molecular Physiology, Penn State Hershey Medical
18 19 20
Running title: Lysosomal exocytosis and Zn2+ toxicity
21 22
To whom correspondence should be addressed: Kirill Kiselyov, Department of Biological
23
Sciences, University of Pittsburgh, 519 Langley Hall, 4249 Fifth Avenue, Pittsburgh, PA, 15260.
24
Tel: 412-624-4317; Fax: 412-624-4759; E-mail: [email protected].
25 26
Key words: Zinc, Golgi, lysosomes, metallothionein, exocytosis, Zn2+ transport, ZnT, Slc30a.
27 28
Word count (excluding references): 6421
29
Figures: 9
30
Supplementary Figures: 3
31 32
JCS Advance Online Article. Posted on 14 May 2014
Journal of Cell Science
Accepted manuscript
Kukic et al., Page 2 of 33
33
Summary
34
Zinc (Zn2+) is an essential micronutrient and an important ionic signal, whose excess as well as
35
scarcity are detrimental to cells. Free cytoplasmic Zn2+ is controlled by a network of Zn2+
36
transporters and chelating proteins. Recently, lysosomes became the focus of studies in Zn2+
37
transport, as they were shown to play a role in zinc-induced toxicity by serving as Zn2+ sinks that
38
absorb Zn2+ from the cytoplasm. Here we investigate the impact of the lysosomal Zn2+ sink on
39
the net cellular Zn2+ distribution and its role in cell death. We found that lysosomes play a
40
cytoprotective role during exposure to extracellular Zn2+. Such a role required lysosomal
41
acidification and exocytosis. Specifically, we found that the inhibition of lysosomal acidification
42
using Bafilomycin A1 (Baf) lead to a redistribution of Zn2+ pools, and increased apoptosis.
43
Additionally, the inhibition of lysosomal exocytosis through knockdown (KD) of the lysosomal
44
SNARE proteins VAMP7 and Synaptotagmin VII (SYT7) suppressed Zn2+ secretion and
45
VAMP7 KD cells had increased apoptosis. These data show that lysosomes play a central role in
46
Zn2+ handling, suggesting a novel Zn2+ detoxification pathway.
47 48
Introduction
49
Cellular Zn2+ dyshomeostasis has been linked to a number of human pathologies including
50
growth defects (Prasad, 2013), impaired immune function (Rink and Gabriel, 2000), diabetes
51
(Jansen et al., 2009), and neurodegenerative diseases (Forsleff et al., 1999; Rulon et al., 2000;
52
Lee et al., 2002; Vinceti et al., 2002). Regulation of cellular Zn2+ levels involves controlling its
53
influx, export and chelation. In general, Zn2+ transport is regulated by ZnT and ZIP transporters,
54
and it is chelated by Zn2+ binding metallothioneins (MTs).
55 56
In addition to Zn2+ evacuation across the plasma membrane (PM) by the Zn2+ transporter ZnT1
57
(Palmiter and Findley, 1995), Zn2+ is exported from the cytoplasm into the organelles by the
58
dedicated ZnT transporters such as ZnT6 for the Golgi (Huang et al., 2002), and ZnT2 and ZnT4
59
for the lysosome (Palmiter et al., 1996; Huang and Gitschier, 1997; Falcon-Perez and
60
Dell'Angelica, 2007; McCormick and Kelleher, 2012). This organellar Zn2+ export lowers
61
potentially toxic cytoplasmic Zn2+ concentrations in pathophysiological conditions such as
62
neurodegeneration (Kanninen et al., 2013) and breast cancer (Lopez et al., 2011). Moreover, it
63
provides Zn2+ to organellar processes that require it, such as the maturation of enzymes like the
Kukic et al., Page 3 of 33
64
lysosomal acid sphingomyelinase (Schissel et al., 1996), and the secretion of Zn2+ under normal
65
physiological conditions such as synaptic transmission (Frederickson and Bush, 2001) and
66
lactation (Kelleher et al., 2009)
Journal of Cell Science
Accepted manuscript
67 68
The upregulation of Zn2+ chelation and transport machinery following the activation of the
69
transcription factor MTF-1 by Zn2+ binding (Andrews, 2001) requires time for transcription,
70
translation and protein processing. It is tempting to speculate that Zn2+ export into organelles
71
serves as a first line of defense to provide temporary Zn2+ storage, giving cells time to upregulate
72
Zn2+ chelators and transporters. Our recent data on the role of lysosomes in Zn2+ handling, as
73
well as some recently published results suggest that lysosomes play a role of such Zn2+ sinks,
74
temporarily storing Zn2+ (Hwang et al., 2008; Kukic et al., 2013). In this paper, we sought to
75
delineate the role of lysosomes in protection against Zn2+-induced toxicity.
76 77
Zn2+ is transported from the cytoplasm into lysosomes by ZnT2 and ZnT4 (Palmiter et al., 1996;
78
Huang and Gitschier, 1997; Falcon-Perez and Dell'Angelica, 2007). Zn2+ can also be delivered to
79
the lysosomes through endocytosis or autophagy (Lee and Koh, 2010; Cho et al., 2012). What
80
happens to Zn2+ absorbed by the lysosomes? A recent series of work from several labs indicate
81
that Zn2+ buildup in the lysosomes is toxic. It leads to lysosomal membrane permeabilization
82
(LMP), to the release of the lysosomal enzymes such as Cathepsins and to cell death (Hwang et
83
al., 2008; Chung et al., 2009; Lee et al., 2009; Hwang et al., 2010). As such, the lysosomal Zn2+
84
accumulation may constitute a cell death mechanism during normal remodeling of Zn2+-rich
85
tissues, such as the mammary gland (Kelleher et al., 2011), as well as in pathological conditions.
86
With this in mind, we sought to answer whether or not the accumulation of Zn2+ in the lysosome
87
is the terminal depot for cellular Zn2+.
88 89
Alternatively, it is possible that lysosomal Zn2+ dissipates and lysosomes constitute only a
90
temporary Zn2+ storage site. Our recently published data suggest that the lysosomal ion channel
91
transient receptor potential mucolipin 1 (TRPML1) is at least partly responsible for dissipating
92
the lysosomal Zn2+ into the cytoplasm (Kukic et al., 2013). It should be noted that lysosomes fuse
93
with the PM via a process involving a specific SNARE complex, which includes the VAMP7
94
protein and SYT7 (Martinez-Arca et al., 2000; Braun et al., 2004; Rao et al., 2004; Logan et al.,
Kukic et al., Page 4 of 33
95
2006; Mollinedo et al., 2006). Such secretion was recently proposed to contribute to excretion of
96
undigested/indigestible products inside lysosomes (Medina et al., 2011). In the course of the
97
present studies, we used VAMP7 and SYT7 KD to suppress lysosomal secretion and assess its
98
role in Zn2+ clearance from the cells.
Journal of Cell Science
Accepted manuscript
99 100
Here we aimed to establish the functional context of the lysosomal Zn2+ accumulation. Our
101
findings indicate that lysosomes actively absorb Zn2+ and secrete it across the PM, since
102
suppressing the lysosomal Zn2+ absorption or secretion causes Zn2+ buildup in the cytoplasm,
103
Golgi apparatus and mitochondria, leading to apoptosis.
104 105
Results
106
Towards testing the role of the lysosomal Zn2+ sink on cellular Zn2+ handling, we blocked the
107
lysosomal H+ pump in HeLa cells using 1 μM Baf and exposed cells to 100 μM ZnCl2 for 3
108
hours. The resulting cytoplasmic Zn2+ spikes were measured using live-cell confocal microscopy
109
and FluoZin-3,AM as described before (Kukic et al., 2013). Figure 1A (Fig 1A) shows that the
110
exposure of Baf-treated cells to Zn2+ caused a significantly higher FluoZin-3,AM response than
111
the exposure of untreated cells to Zn2+. Although Baf has been shown to decrease cytoplasmic
112
pH, potentially affecting Zn2+ binding to cytoplasmic proteins, or FluoZin-3,AM fluorescence,
113
the magnitude of the observed effects appear to be incompatible with the quantitative estimates
114
of changes induced by Baf. Thus, Baf’s effect on cytoplasmic pH appears to be small, within
115
only tenths of pH units (Heming et al., 1995). The degree of pH change necessary to cause an
116
effect on Zn2+ handling, on the other hand, significantly exceeds that reported to be caused by
117
Baf. A pH drop below 6.7 is required to trigger an increase in intracellular Zn2+ according to one
118
set of studies (Kiedrowski, 2012), while another set showed that metallothioneins release Zn2+
119
only after cytoplasmic pH drops below 5.0 (Jiang et al., 2000). Thus, the increase in cytoplasmic
120
Zn2+ caused by Baf likely correlates with the loss of lysosomal function, rather than cytosolic pH
121
changes.
122 123
We have previously shown that Zn2+ transporters ZnT2 and ZnT4 colocalize with the lysosomal
124
ion channel TRPML1 in HeLa cells (Kukic et al., 2013). We suggested that these transporters
125
play a role in loading of the lysosomes with Zn2+. In order to test this assumption, we KD ZnT2
Kukic et al., Page 5 of 33
126
and ZnT4 using siRNA as described before and tested the resulting changes in Zn2+ handling
127
using FluoZin-3,AM. Fig 1B shows that ZnT2 and ZnT4 KD increased cytoplasmic Zn2+ levels
128
observed in these cells after 3 hour long treatment with 100 μM ZnCl2. These results are in
129
agreement with the previously published data on the dependence of ZnTs activity on the acidic
130
environment of the lysosomes (Chao and Fu, 2004; Ohana et al., 2009) for Zn2+ binding and
131
transporting activity.
Journal of Cell Science
Accepted manuscript
132 133
The upregulation of MTF-1–dependent, Zn2+-responsive genes such as MT2a and ZnT1 (Saydam
134
et al., 2002) indicates elevated cytoplasmic Zn2+. MT2a mRNA was used previously in our
135
studies of the role of TRPML1 in Zn2+ handling. We measured the expression of the mRNA of
136
these genes using qRT-PCR (Fig 2). An increase in MT2a and ZnT1 mRNA responses to Zn2+ in
137
cells treated with Baf is evident. With MT2a mRNA levels in DMSO-treated (no Zn2+) cells
138
taken for 100%, MT2a mRNA levels were 816.39±73.61% in cells treated with DMSO+Zn2+
139
(n=4; p
Kukic et al., Page 1 of 33
1 2 3 4 5 6 7
Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity.
8
Journal of Cell Science
Accepted manuscript
9
Ira Kukic1, Shannon L. Kelleher2,3,4 and Kirill Kiselyov1
10 11 12
1
13
15260 USA,
14
Development, The Pennsylvania State University, University Park, Pennsylvania 16802, USA,
15
Department of Surgery, Penn State Hershey Medical Center, Hershey, Pennsylvania 17033,
16
USA, and
17
Center, Hershey, Pennsylvania 17033, USA.
From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania
4
2
the Department if Nutritional Sciences, College of Health and Human 3
Department of Cellular and Molecular Physiology, Penn State Hershey Medical
18 19 20
Running title: Lysosomal exocytosis and Zn2+ toxicity
21 22
To whom correspondence should be addressed: Kirill Kiselyov, Department of Biological
23
Sciences, University of Pittsburgh, 519 Langley Hall, 4249 Fifth Avenue, Pittsburgh, PA, 15260.
24
Tel: 412-624-4317; Fax: 412-624-4759; E-mail: [email protected].
25 26
Key words: Zinc, Golgi, lysosomes, metallothionein, exocytosis, Zn2+ transport, ZnT, Slc30a.
27 28
Word count (excluding references): 6421
29
Figures: 9
30
Supplementary Figures: 3
31 32
JCS Advance Online Article. Posted on 14 May 2014
Journal of Cell Science
Accepted manuscript
Kukic et al., Page 2 of 33
33
Summary
34
Zinc (Zn2+) is an essential micronutrient and an important ionic signal, whose excess as well as
35
scarcity are detrimental to cells. Free cytoplasmic Zn2+ is controlled by a network of Zn2+
36
transporters and chelating proteins. Recently, lysosomes became the focus of studies in Zn2+
37
transport, as they were shown to play a role in zinc-induced toxicity by serving as Zn2+ sinks that
38
absorb Zn2+ from the cytoplasm. Here we investigate the impact of the lysosomal Zn2+ sink on
39
the net cellular Zn2+ distribution and its role in cell death. We found that lysosomes play a
40
cytoprotective role during exposure to extracellular Zn2+. Such a role required lysosomal
41
acidification and exocytosis. Specifically, we found that the inhibition of lysosomal acidification
42
using Bafilomycin A1 (Baf) lead to a redistribution of Zn2+ pools, and increased apoptosis.
43
Additionally, the inhibition of lysosomal exocytosis through knockdown (KD) of the lysosomal
44
SNARE proteins VAMP7 and Synaptotagmin VII (SYT7) suppressed Zn2+ secretion and
45
VAMP7 KD cells had increased apoptosis. These data show that lysosomes play a central role in
46
Zn2+ handling, suggesting a novel Zn2+ detoxification pathway.
47 48
Introduction
49
Cellular Zn2+ dyshomeostasis has been linked to a number of human pathologies including
50
growth defects (Prasad, 2013), impaired immune function (Rink and Gabriel, 2000), diabetes
51
(Jansen et al., 2009), and neurodegenerative diseases (Forsleff et al., 1999; Rulon et al., 2000;
52
Lee et al., 2002; Vinceti et al., 2002). Regulation of cellular Zn2+ levels involves controlling its
53
influx, export and chelation. In general, Zn2+ transport is regulated by ZnT and ZIP transporters,
54
and it is chelated by Zn2+ binding metallothioneins (MTs).
55 56
In addition to Zn2+ evacuation across the plasma membrane (PM) by the Zn2+ transporter ZnT1
57
(Palmiter and Findley, 1995), Zn2+ is exported from the cytoplasm into the organelles by the
58
dedicated ZnT transporters such as ZnT6 for the Golgi (Huang et al., 2002), and ZnT2 and ZnT4
59
for the lysosome (Palmiter et al., 1996; Huang and Gitschier, 1997; Falcon-Perez and
60
Dell'Angelica, 2007; McCormick and Kelleher, 2012). This organellar Zn2+ export lowers
61
potentially toxic cytoplasmic Zn2+ concentrations in pathophysiological conditions such as
62
neurodegeneration (Kanninen et al., 2013) and breast cancer (Lopez et al., 2011). Moreover, it
63
provides Zn2+ to organellar processes that require it, such as the maturation of enzymes like the
Kukic et al., Page 3 of 33
64
lysosomal acid sphingomyelinase (Schissel et al., 1996), and the secretion of Zn2+ under normal
65
physiological conditions such as synaptic transmission (Frederickson and Bush, 2001) and
66
lactation (Kelleher et al., 2009)
Journal of Cell Science
Accepted manuscript
67 68
The upregulation of Zn2+ chelation and transport machinery following the activation of the
69
transcription factor MTF-1 by Zn2+ binding (Andrews, 2001) requires time for transcription,
70
translation and protein processing. It is tempting to speculate that Zn2+ export into organelles
71
serves as a first line of defense to provide temporary Zn2+ storage, giving cells time to upregulate
72
Zn2+ chelators and transporters. Our recent data on the role of lysosomes in Zn2+ handling, as
73
well as some recently published results suggest that lysosomes play a role of such Zn2+ sinks,
74
temporarily storing Zn2+ (Hwang et al., 2008; Kukic et al., 2013). In this paper, we sought to
75
delineate the role of lysosomes in protection against Zn2+-induced toxicity.
76 77
Zn2+ is transported from the cytoplasm into lysosomes by ZnT2 and ZnT4 (Palmiter et al., 1996;
78
Huang and Gitschier, 1997; Falcon-Perez and Dell'Angelica, 2007). Zn2+ can also be delivered to
79
the lysosomes through endocytosis or autophagy (Lee and Koh, 2010; Cho et al., 2012). What
80
happens to Zn2+ absorbed by the lysosomes? A recent series of work from several labs indicate
81
that Zn2+ buildup in the lysosomes is toxic. It leads to lysosomal membrane permeabilization
82
(LMP), to the release of the lysosomal enzymes such as Cathepsins and to cell death (Hwang et
83
al., 2008; Chung et al., 2009; Lee et al., 2009; Hwang et al., 2010). As such, the lysosomal Zn2+
84
accumulation may constitute a cell death mechanism during normal remodeling of Zn2+-rich
85
tissues, such as the mammary gland (Kelleher et al., 2011), as well as in pathological conditions.
86
With this in mind, we sought to answer whether or not the accumulation of Zn2+ in the lysosome
87
is the terminal depot for cellular Zn2+.
88 89
Alternatively, it is possible that lysosomal Zn2+ dissipates and lysosomes constitute only a
90
temporary Zn2+ storage site. Our recently published data suggest that the lysosomal ion channel
91
transient receptor potential mucolipin 1 (TRPML1) is at least partly responsible for dissipating
92
the lysosomal Zn2+ into the cytoplasm (Kukic et al., 2013). It should be noted that lysosomes fuse
93
with the PM via a process involving a specific SNARE complex, which includes the VAMP7
94
protein and SYT7 (Martinez-Arca et al., 2000; Braun et al., 2004; Rao et al., 2004; Logan et al.,
Kukic et al., Page 4 of 33
95
2006; Mollinedo et al., 2006). Such secretion was recently proposed to contribute to excretion of
96
undigested/indigestible products inside lysosomes (Medina et al., 2011). In the course of the
97
present studies, we used VAMP7 and SYT7 KD to suppress lysosomal secretion and assess its
98
role in Zn2+ clearance from the cells.
Journal of Cell Science
Accepted manuscript
99 100
Here we aimed to establish the functional context of the lysosomal Zn2+ accumulation. Our
101
findings indicate that lysosomes actively absorb Zn2+ and secrete it across the PM, since
102
suppressing the lysosomal Zn2+ absorption or secretion causes Zn2+ buildup in the cytoplasm,
103
Golgi apparatus and mitochondria, leading to apoptosis.
104 105
Results
106
Towards testing the role of the lysosomal Zn2+ sink on cellular Zn2+ handling, we blocked the
107
lysosomal H+ pump in HeLa cells using 1 μM Baf and exposed cells to 100 μM ZnCl2 for 3
108
hours. The resulting cytoplasmic Zn2+ spikes were measured using live-cell confocal microscopy
109
and FluoZin-3,AM as described before (Kukic et al., 2013). Figure 1A (Fig 1A) shows that the
110
exposure of Baf-treated cells to Zn2+ caused a significantly higher FluoZin-3,AM response than
111
the exposure of untreated cells to Zn2+. Although Baf has been shown to decrease cytoplasmic
112
pH, potentially affecting Zn2+ binding to cytoplasmic proteins, or FluoZin-3,AM fluorescence,
113
the magnitude of the observed effects appear to be incompatible with the quantitative estimates
114
of changes induced by Baf. Thus, Baf’s effect on cytoplasmic pH appears to be small, within
115
only tenths of pH units (Heming et al., 1995). The degree of pH change necessary to cause an
116
effect on Zn2+ handling, on the other hand, significantly exceeds that reported to be caused by
117
Baf. A pH drop below 6.7 is required to trigger an increase in intracellular Zn2+ according to one
118
set of studies (Kiedrowski, 2012), while another set showed that metallothioneins release Zn2+
119
only after cytoplasmic pH drops below 5.0 (Jiang et al., 2000). Thus, the increase in cytoplasmic
120
Zn2+ caused by Baf likely correlates with the loss of lysosomal function, rather than cytosolic pH
121
changes.
122 123
We have previously shown that Zn2+ transporters ZnT2 and ZnT4 colocalize with the lysosomal
124
ion channel TRPML1 in HeLa cells (Kukic et al., 2013). We suggested that these transporters
125
play a role in loading of the lysosomes with Zn2+. In order to test this assumption, we KD ZnT2
Kukic et al., Page 5 of 33
126
and ZnT4 using siRNA as described before and tested the resulting changes in Zn2+ handling
127
using FluoZin-3,AM. Fig 1B shows that ZnT2 and ZnT4 KD increased cytoplasmic Zn2+ levels
128
observed in these cells after 3 hour long treatment with 100 μM ZnCl2. These results are in
129
agreement with the previously published data on the dependence of ZnTs activity on the acidic
130
environment of the lysosomes (Chao and Fu, 2004; Ohana et al., 2009) for Zn2+ binding and
131
transporting activity.
Journal of Cell Science
Accepted manuscript
132 133
The upregulation of MTF-1–dependent, Zn2+-responsive genes such as MT2a and ZnT1 (Saydam
134
et al., 2002) indicates elevated cytoplasmic Zn2+. MT2a mRNA was used previously in our
135
studies of the role of TRPML1 in Zn2+ handling. We measured the expression of the mRNA of
136
these genes using qRT-PCR (Fig 2). An increase in MT2a and ZnT1 mRNA responses to Zn2+ in
137
cells treated with Baf is evident. With MT2a mRNA levels in DMSO-treated (no Zn2+) cells
138
taken for 100%, MT2a mRNA levels were 816.39±73.61% in cells treated with DMSO+Zn2+
139
(n=4; p
