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,1
* Department of Cell Biology, Institute of Anatomy, University of Bern, Switzerland;
Institute of Physiology, Taras Schevchenko Kiev University, Ukraine;
Department of Urology, University of Bern, Switzerland; and
The Physiological Laboratory, University of Liverpool, Liverpool, UK
2Correspondence: Department of Cell Biology, Institute of Anatomy, University of Bern, Bühlstr. 26, 3012 Bern, Switzerland. E-mail: draeger{at}ana.unibe.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: 5'-nucleotidase • microdomains • contraction • protein kinase C • RhoA • compartmentalization • annexins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. In addition to these so-called macrodomains, the sarcolemma of smooth muscle is further subdivided into ‘microdomains’ of glycerolipid-containing non-raft regions and glycolipid- and cholesterol-enriched membrane rafts (1
, 3)
. Lipid rafts can be classified into ‘caveolar’ and ‘non-caveolar’ types (4
, 5)
. The former is stationary and enhances the stability of associated receptor molecules, whereas the latter is presumed to be a smaller, mobile, and transitory unit. Since the reversible association of a particular protein with a distinct lipid microdomain is essential for activation of its downstream effectors, regulation of the lateral movement of these structures and their temporary or permanent association with the plasma membrane are of paramount importance for optimal functioning. We have recently shown that annexin 2 stabilizes smooth muscle microdomain-structure by promoting the Ca2+-dependent association of membrane rafts (3)
. Here, we have identified a mechanism whereby annexin 2-raft binding can be interrupted and assume that an identical process applies for the interaction of annexin 1 with non-raft regions. This annihilation of raft binding involves protein cleavage by the membrane-associated protease calpain. Since the truncated protein is incapable of binding to the cell membrane or sustaining its spatial segregation, calpain-induced cleavage may well terminate the signaling event. This cleavage, which is dependent on a fully functional contractile apparatus, is tissue and protein specific. That it is observed in neither endothelial nor HeLa cells points to a differential regulation of membrane signaling in different cell types.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pieces of smooth muscle were excised from the human bladder during surgical procedures that were unrelated to muscle disease. With the aid of a light microscope, they were teased into individual bundles. Processing for contraction/relaxation experiments, ultracryomicrotomy, and immunolabeling were performed as described previously (2)
.
Immunohistochemistry and antibodies
Immunolabeling was performed as described by Jostarndt-Fögen et al. (6)
. Monoclonal antibodies against annexins 1, 2, 4, and 6 and a polyclonal one against caveolin were purchased from Transduction Laboratories (Lexington, KY). A monoclonal antibody against vinculin (V9131) was obtained from Sigma (Buchs, Switzerland); a monoclonal antibody against RhoA and a polyclonal one against PKC
were from Santa Cruz. For dual-labeling purposes, a polyclonal antibody against annexin 2 was generated in a rabbit (3)
.
Fluorescent labeling was performed using Cy3 (Jackson, Baltimore, MD) or Alexa-conjugated (Molecular Probes, Eugene, OR) secondary antibodies. Negative controls were generated by either absorbing the antibody with purified antigen (for annexins 2 and 6) or applying a nonbinding primary antibody.
Tissue sections were examined in a Zeiss Axiophot fluorescent microscope and images were collected using a digital CCD camera (Ultrapix, Astrocam).
Isolation of smooth muscle microsomal and detergent-resistant glycolipid-enriched (DIG) membranes
Microsomal membranes were isolated from porcine stomach smooth muscle or cultured human myometrial cells according to the protocol described previously (3)
. Unless otherwise stated, all procedures were performed at 4°C or on ice. Minced muscle or scraped and pelleted cells were routinely extracted in 5 volumes of buffer A (60 mM KCl, 2 mM MgCl2, 0.2 mM CaCl2, and 20 mM imidazol; pH 7.0). When indicated, the extraction was performed in the presence of 2 mM EGTA. After low-speed centrifugation at 10'000 g for 30 min, the supernatant was centrifuged at high speed (50'000 g) for 90 min. The pellets containing the microsomal membranes were washed three times (with intervening centrifugations at 10'000 g for 30 min) and resuspended in 5 volumes of buffer B (120 mM KCl, 0.2 mM CaCl2, and 20 mM imidazol; pH 7.0). The DIG membrane fraction was obtained by following a procedure similar to that described above for microsomal membranes; the extraction step in this case was performed in the presence of a non-ionic detergent (0.5% Nonidet P-40).
Extraction of microsomal membranes with Nonidet P-40
Aliquots (100 µL) of the microsomal membrane preparations (protein concentration=4 mg/mL) were incubated for 10 min on ice in buffer B containing different concentrations of Nonidet P-40. The suspensions were centrifuged at low speed (10,000 g for 30 min) and the resulting supernatants and pellets (made up to 100 µL) were analyzed by Western blotting.
EGTA extraction of microsomal membranes, turbidity measurements, and annexin binding.
A 100 mM solution of EGTA (10 µL) was added to 990 µl aliquots of smooth muscle microsomal membrane preparations (suspended in buffer B; protein concentration=4 mg/mL). After a 10 min incubation period at ambient temperature, the suspensions were centrifuged at 10,000 g for 30 min. Aliquots (1.25 µL) of a 20 mM solution of CaCl2 were added stepwise (with intervening 2 min incubations at ambient temperature) to 500 µL of the resulting supernatant. The Ca2+-dependent increase in turbidity was spectrophotometrically measured at 500 nm (A500). After the final addition of CaCl2, the suspension was centrifuged (10,000 g for 30 min) and the resulting supernatant and pellet (made up to 500 µL) was analyzed by Western blotting.
Endogenous cleavage of annexins in cultured cells
Primary cultures of human myometrial cells (7)
and human microvascular endothelial cells (passages 2–5) (Promocell, Heidelberg, Germany) or HeLa cells [passage unknown (ATCC, Rockville, MD)] were grown to various degrees of confluency on plastic Petri dishes (10 cm Ø). Unless indicated otherwise, densely confluent cultures were used for all experiments. The attached cells were washed extensively with Na+-Tyrode’s solution containing 2 mM CaCl2 and released from their substrate by gentle scraping. They were permitted to contract in 200 µL of Na+-Tyrode’s solution for specified intervals. At the end of an experiment, lysis was induced by adding 100 µL of SDS sample buffer to the cell suspension. In control experiments, the attached cells were lysed by adding 200 µL of 9M urea, then 100 µL of the SDS sample buffer. When indicated, the attached cells were incubated for specified periods in Na+-Tyrode’s solution containing defined concentrations of the various reagents.
Miscellaneous
5'-Nucleotidase activity was measured according to a modified version of the method published by Fiske and Subarrow (8)
using 5'-AMP as a substrate (2)
. Thin-layer chromatography was performed basically in the manner described by Macala et al. (9)
with a few modifications (2)
. SDS-PAGE was carried out using the procedure published by Laemmli (10)
. Blotting of gels onto immobilon-P membranes (Millipore Corporation, Bedford, MA) was conducted as detailed by Towbin et al. (11)
. Immunoreactivity was detected using a secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, UK) and visualized by enhanced chemoluminescence (Amersham Pharmacia Biotech, UK). Protein concentrations were determined according to the method described by Bradford (12)
using bovine serum albumin as a standard. Free Ca2+ concentrations ([Ca2+]free) were estimated using MAX CHELATOR software (Chris Patton, Stanford University, Hopkins Marine Station). Western blots were scanned and analyzed using IMAGEQUANT 3.3 software (Molecular Dynamics, Amersham Pharmacia Biotech, Sweden).
A specific cell-permeable inhibitor of calpain (MDL 28170) was purchased from Calbiochem (San Diego, CA) and diluted in di-methyl-sulfoxide (DMSO). It was added to the culture medium to yield a final concentration of 30 µM (13)
. All data shown are representative of at least three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cleavage of annexins 1 and 2
In culture, smooth muscle cells adhere to a rigid substratum and are unable to shorten even in the presence of contractile agonists. Although smooth muscle-specific protein isoforms are down-regulated in such cells, they nevertheless develop an extensive actin cytoskeleton, produce static force, and are still capable of contraction (14)
when released from their substrate by gentle scraping. This mechanical detachment, accompanied by cellular contraction and a fundamental rearrangement of sarcolemma and cytoskeleton, results in rapid but limited cleavage of annexin 2 and complete degradation of annexin 1 (Fig. 1
a). The cleavage occurs on the cell membrane (see below) and is inhibited by MDL, a specific inhibitor of calpain, indicating that this protease is responsible for the proteolysis (Fig. 1b
). Other proteins associated with the smooth muscle cell membrane, such as vinculin and caveolin as well as annexins 4 and 6, remain intact (Fig. 1c
), suggesting that the observed proteolysis is highly specific.
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Cellular contraction involves a cyclic sliding of force-generating myosin over anchored actin filaments, the movement being triggered by phosphorylation of the regulatory myosin light chains and mediated by a Ca2+/calmodulin-dependent myosin light chain kinase (15)
. If this system is interfered with by inhibiting actin polymerization (with cytochalasin D) or the myosin light chain kinase (with wortmannin), annexin 2 truncation (Fig. 2
a–c) or annexin 1 degradation is suppressed.
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The transition from a ‘synthetic’ to a ‘contractile’ phenotype in culture is triggered when the cells reach confluence (16)
. Proteolysis of annexins 1 (not shown) and 2 is much more pronounced in densely than in barely confluent cultures (Fig. 2d
), thus supporting our suggestion that contraction is a prerequisite for this phenomenon. Indeed, the cleavage occurred solely in smooth muscle cells, not being observed in either primary cultures of endothelial or HeLa cells (not shown), which do not possess a well-developed contractile apparatus.
Smooth muscle cell contraction is elicited by increasing concentrations of intracellular Ca2+ ([Ca2+]i), and the cleavage of annexins 1 and 2 depended critically on it. When Ca2+ was removed from the extracellular milieu by adding EGTA or its entry was blocked by introducing nonselective inorganic Ca2+ antagonists, cleavage was completely inhibited (Fig. 2e
). However, whereas intracellular Ca2+ was found to be essential for cleavage, a rise in its concentration elicited by exposure of substratum-attached cells to contractile agonists (60 mM KCl, 10 nM angiotensin II, 100 µM ATP) or by treatment with Ca2+-ionophore (2 µM ionomycin) did not in itself set off protein cleavage (not shown). This observation implies that contraction itself constitutes the trigger for proteolysis.
In transverse sections through a smooth muscle bundle of the urinary bladder wall, antibodies against annexins 6 and 2 associate with the sarcolemma in contracted cells whereas diffuse cytoplasmic labeling can be observed in relaxed ones (Fig. 3
a, b).
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These results are corroborated by the association of annexins with plasma membrane preparations (microsomes) obtained from porcine stomach smooth muscle tissue in the presence of micromolar [Ca2+]free (3)
. In the microsomes thus obtained, both annexin 1 and annexin 2, but not annexins 4 and 6, were present in their native and truncated forms (Fig. 4
). The fact that no truncation of annexins was observed in similar microsomal preparations originating from structurally and functionally related striated muscle tissues (not shown) points to the existence of a pathway for annexin cleavage that is specific for smooth muscle cells.
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The sarcolemma of smooth muscle cells encompasses a highly dynamic microdomain structure consisting of glycolipid- and cholesterol-enriched rafts that are spatially segregated from non-raft glycerophospholipid regions (3)
. Glycerophospholipids are readily extracted by non-ionic detergents whereas rafts are resistant to this treatment (17)
(Fig. 5
a). In contrast, membrane rafts can be destabilized by selective extraction of cholesterol with methyl-ß-cyclodextrin (18)
. In smooth muscle tissue (not shown) and cultured cells, the truncation of annexins was abolished in the presence of non-ionic detergent Nonidet P-40, yet remained unaffected by methyl-ß-cyclodextrin (Fig. 5b
). Since annexin membrane translocation (Fig. 3)
and protein cleavage require elevated [Ca2+]i (Fig. 2)
and the latter also depended critically on intact glycerophospholipid-containing regions, it is not unreasonable to assume that proteolysis must occur on the sarcolemma at sites that correspond to non-raft domains.
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Annexins are markers for raft and non-raft domains
In an earlier investigation, we demonstrated that different members of the annexin family show preferences for distinct membrane microcompartments (3)
. In the present study, we illustrate that the greater part of annexin 2 is detergent insoluble and raft associated, whereas readily extracted annexin 1 localizes to non-raft regions (Fig. 5c
).
In support of our hypothesis that proteolysis is restricted to non-raft regions, non-raft annexin 1 degraded preferentially, more rapidly, and to a greater extent than raft annexin 2 (Fig. 1)
. Truncation of the latter occurred more slowly, most likely due to the limited rate of its dissociation from rafts and reassociation with non-raft regions. As a result, the amount of truncated annexin 2 (Fig. 1)
correlated with the amount of its non-raft, detergent-soluble fraction (Fig. 5c
).
Only the native—not the cleaved—form of annexin 2 was capable of binding to rafts (Fig. 6
a). Apparently, the latter is also true for the association of degraded annexin 1 with non-raft regions. The truncated form of annexin 2 nevertheless retained its ability to bind to membrane microsomes purified in the absence of detergent (Fig. 5a
), albeit with much lower Ca2+ sensitivity (Fig. 5b
).
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Functional segregation of membrane domains
Our previous work demonstrated that the Ca2+-dependent binding of annexin 2 to membrane rafts elicited their association (3)
. Likewise, the Ca2+-dependent association of membranes containing acidic phospholipids by annexin 1 is well documented (reviewed in refs 19
, 20
). The elevation in [Ca2+]i during smooth muscle contraction might thus result in an increased segregation of membrane constituents brought about by annexin-dependent associations directed against different membrane microcompartments. The partitioning of 5'-nucleotidase (3)
, protein kinase C (PKC), and RhoA—major signaling molecules responsible for the modulation of smooth muscle contraction (reviewed in refs 15
, 21
)—into distinct membrane microdomains (Fig. 7
) provides additional evidence for the importance of membrane segregation in the regulation of smooth muscle contractility.
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This observation raises the possibility that during regular contraction-relaxation events, the activities and/or distribution of 5'-nucleotidase, RhoA, and PKC might be modulated in an annexin- and Ca2+-dependent manner via their association with distinct microdomains. Our findings also suggest that the cleavage of annexins 1 and 2 by calpain can be triggered by contractile events that exceed the normal stress response of smooth muscle cells or occur during smooth muscle remodeling. This event abolishes binding of the annexins to their membrane counterparts and thereby might interrupt segregation-mediated signaling (Fig. 8
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 23)
and to regulate the intracellular localization of these proteins (22)
. Amino-terminal cleavage of annexins 1 and 2 has been observed in both neutrophils (24)
and pulmonary epithelial cells (25
, 26)
and has been assigned a role in endocyctic vesicle maturation and secretion (25
, 27
, 28)
. This phenomenon has been variously attributed to enzymatic degradation by either a metalloprotease (28)
, cathepsin (22)
, calpain (25)
, or to chaperone-mediated autophagy (29)
.
We now report that annexin 1 undergoes complete (and annexin 2, partial) proteolysis in smooth muscle cells. After blocking of calpain activity with the cell-permeable inhibitor (MDL; 13
), proteolysis of annexins 1 and 2 was significantly inhibited, indicating that in smooth muscle, calpain constitutes a potential candidate for their cleavage.
Annexins 4 and 6 were not susceptible to proteolysis, indicating significant structural differences between these two groups of annexins. In contrast to annexins 4 and 6, annexins 1 and 2 are known to form heterotetramers (reviewed in ref 20
). The NH2 terminus of annexin 2 has been shown to be essential for tetramer formation and harbors phosphorylation sites for pp60 src and protein kinase C (reviewed in ref 20
). That the truncated form of annexin 1 could be generated (Fig. 4)
suggests that the differences in the proteolysis pattern between annexins 1 and 2 result from a higher degree of annexin 1 proteolysis but not, or to a lesser extent, from structural differences between these two proteins.
In a previous study we demonstrated that selected members of the annexin family interact preferentially with different lipid binding partners (3)
. These data appeared to be incompatible with numerous reports, indicating that all annexins display a distinct preference for acidic glycerophospholipids (reviewed in (19)
. The results of the present investigation now permit a reconciliation of these apparently disparate findings: in the presence of Ca2+, the core domain of annexin 2 associates with glycerolipids whereas its amino-terminal domain enables the molecule to interact with glycolipid- and cholesterol-enriched rafts. A cholesterol-dependent association of annexin 2 with early endosomes has been shown to depend on the presence of amino acids 15 to 24 of the unique terminal domain of annexin 2 (30
, 31)
. It is likely that the calpain-mediated truncation observed in our study results in loss of precisely the same amino-terminal domain.
Cleavage of annexins 1 and 2 occurs only in contractile cells
After mechanical detachment, the vast majority of cultured cells remained impermeable to Trypan blue (data not shown), which indicates that by and large plasmalemmal integrity was not compromised by this treatment. That antagonists of Ca2+ influx likewise inhibited proteolysis furnishes additional evidence of plasmalemmal integrity in cultured cells released by mechanical scraping. Whereas intracellular Ca2+ was found to be essential for cleavage, a rise in its concentration [elicited by 60 mM KCl, 10 nM angiotensin II, 100 µM ATP, or treatment with 2 µM ionomycin (not shown)] did not suffice to trigger the proteolysis of annexin 2 in myometrial cells not subjected to mechanical detachment.
Interference with the cell’s contractile activity by disassemblage of the actin cytoskeleton by cytochalasin D or inhibition of myosin light chain kinase by wortmannin significantly reduced proteolysis (see Fig. 3
). Neither endothelial nor HeLa cells possess an extensive actin cytoskeleton. In neither cell type did annexins 1 and 2 undergo proteolysis after their mechanical detachment from the culture substratum. Similarly, in nonconfluent smooth muscle cells, which lack a dense meshwork of stress fibers and express but weakly the -smooth muscle actin isoform, no significant truncation of annexins 1 and 2 was observed.
Proteolysis—in particular, of annexin 1—was more obvious in cultured cells than in smooth muscle tissue (see Figs. 1
, 4
). Its limited truncation but not total degradation was observed in tissue in contrast to cell culture conditions. This indicates that proteolysis and its related changes in annexin-dependent signaling might be more important for the contractile activity of smooth muscle during cell differentiation and remodeling than for contraction of differentiated tissue. The physiological significance of annexin cleavage for smooth muscle cell function is unknown. An additional mechanism for regulation of membrane segregation during sustained contraction might provide a means to cope with pathological states. Associated with increased smooth muscle workload are common diseases such as urinary bladder obstruction after prostatic hyperplasia or hypertension.
Raft-mediated signaling in smooth muscle cells
Smooth muscle cells do not possess the elaborate, stretch-sensing systems of skeletal muscle; nevertheless, their plasmalemma has to adjust to ever-varying changes in their length and shape during contraction-relaxation cycles and during developmental remodeling. Given the paucity of their innervation and the sparsity of their intercellular connections, it is reasonable to deduce that the mechanical transduction of forces responsible for these shape changes might contribute to the organization of smooth muscle contractile apparatus and cytoskeleton. The F-actin binding site of annexin 2 has recently been identified (32)
and a direct link between raft clustering and the actin cytoskeleton has been established (33)
.
The importance of raft-dependent signaling is well established in many biological processes, especially in regulation of the immune responses (34
, 35)
. In a recent publication (3)
, we proposed that the Ca2+-dependent binding of annexin 2 to membrane lipids could lead to association of rafts and ensuing segregation of membrane microcompartments, thus modulating signaling events mediated by these microdomains. Here we suggest that in addition to annexin 2-dependent raft-association, the interaction between annexin 1 and non-raft regions could play a role in the segregation of membrane lipids. According to our concept, an initial signal generated after the cell’s stimulation might be modulated via an annexin-dependent compartmentalization of the cell membrane and subsequent local changes in the physicochemical properties of the membranous lipid bilayer. In smooth muscle, such segregation is most likely regulated via fluctuations in [Ca2+]free (3)
. Cleavage of annexins 1 and 2 with subsequent loss of association with their respective membrane microcompartment might represent an additional means—with presumably longer lasting and more dramatic consequences—to interrupt this signaling pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 15, 2002.
Revision received April 15, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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