Calpain Expression and Activity during Lens Fiber Cell
Differentiation |
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Authors: | Alicia De Maria Yanrong Shi Nalin M Kumar and Steven Bassnett |
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Institution: | ‡Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110 and the §Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois 60612 |
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Abstract: | In animal models, the dysregulated activity of calcium-activated proteases,
calpains, contributes directly to cataract formation. However, the
physiological role of calpains in the healthy lens is not well defined. In
this study, we examined the expression pattern of calpains in the mouse lens.
Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7
were expressed in lens fiber cells. Using controlled lysis, depth-dependent
expression profiles for each calpain were obtained. These indicated that,
unlike calpain 1, 2, and 7, which were most abundant in cells near the lens
surface, calpain 3 expression was strongest in the deep cortical region of the
lens. We detected calpain activities in vitro and showed that
calpains were active in vivo by microinjecting fluorogenic calpain
substrates into cortical fiber cells. To identify endogenous calpain
substrates, membrane/cytoskeleton preparations were treated with recombinant
calpain, and cleaved products were identified by two-dimensional difference
electrophoresis/mass spectrometry. Among the calpain substrates identified by
this approach was αII-spectrin. An antibody that specifically recognized
calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved
in vivo, late in fiber cell differentiation, at or about the time
that lens organelles are degraded. The generation of the calpain-specific
spectrin cleavage product was not observed in lens tissue from calpain 3-null
mice, indicating that calpain 3 is uniquely activated during lens fiber
differentiation. Our data suggest a role for calpains in the remodeling of the
membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium
dependence of the founder members of the family, the ubiquitously expressed
enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain
family includes more than a dozen members with sequence relatedness to the
catalytic subunits of calpain 1 and 2. Calpains have a modular domain
architecture. By convention, the family is subdivided into classical and
nonclassical calpains, according to the presence or absence, respectively, of
a calcium-binding penta-EF-hand module in domain IV of the protein
(1). Classical calpains include
calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7,
10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an
essential role for calpains during embryonic development. Knock-out of the
small regulatory subunit (Capn4) results in embryonic lethality
(2,
3). Similarly, inactivation of
the Capn2 gene blocks development between the morula and blastocyst
stage (4). In humans, mutations
in CAPN3 underlie limb-girdle muscular dystrophy-2A, and
polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus
(5,
6).Even under conditions of calcium overload, where calpains are presumably
activated maximally, only a subset (<5%) of cellular proteins are
hydrolyzed (7). Calpains
typically cleave their substrates at a limited number of sites to generate
large polypeptide fragments that, in many cases, retain bioactivity. Thus,
under physiological conditions, calpains probably participate in the
regulation of protein function rather than in non-specific protein
degradation.More than 100 proteins have been shown to serve as calpain substrates
in vitro, including cytoskeletal proteins
(8), signal transduction
molecules (9), ion channels
(10), and receptors
(11). In vivo,
calpains are believed to function in myoblast fusion
(12), long term potentiation
(13), and cellular mobility
(14). Unregulated calpain
activity, secondary to intracellular calcium overload, is associated with
several pathological conditions, including Alzheimer disease
(15), animal models of
cataract (16), myocardial
(17), and cerebral ischemia
(18).In addition to their domain structure, calpains are often classified
according to their tissue expression patterns. Calpain 1, 2, and 10 are widely
expressed in mammalian tissues, but other members of the calpain family show
tissue-specific expression patterns. Calpain 8, for example, is a
stomach-specific calpain (19),
whereas expression of calpain 9 is restricted to tissues of the digestive
tract (20). The expression of
calpain 3 was originally thought to be limited to skeletal muscle
(21), but splice variants of
calpain 3 have since been detected in a range of tissues. At least 12 isoforms
of calpain 3 have been described in rodents
(22), of which several are
expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88
(retina) (23).Calpains have been studied intensively in the ocular lens because of their
suspected involvement in lens opacification (cataract). Calpain-mediated
proteolysis of lens crystallin proteins causes increased light scatter
(24). Unregulated activation
of calpains is observed in rodent cataract models
(25), where calpain-mediated
degradation of crystallin proteins
(26) and cytoskeletal elements
(27) is commonly observed.
Calpain inhibitors are effective in delaying or preventing cataract in
vitro (28,
29) and in vivo
(30,
31).It is likely, however, that calpains have important physiological roles in
the lens beyond their involvement in tissue pathology. Terminal
differentiation of lens fiber cells involves a series of profound
morphological and biochemical transformations. For example, differentiating
lens fiber cells undergo an enormous (>100-fold) increase in cell length,
accompanied by extensive remodeling of the plasma membrane system
(32). Early in the
differentiation process, fusion pores are established between cells, as
neighboring fibers are incorporated into the lens syncytium
(33). A later stage of fiber
cell differentiation involves the dissolution of all intracellular organelles,
a process that is thought to eliminate light-scattering particles from the
light path and contribute to the transparency of the tissue
(34). Any or all of these
phenomena might require the developmentally regulated activation of calpains.
This is consistent with our previous observation that in calpain 3 knock-out
mice the transition zone is altered, suggesting a change in the
differentiation program
(35).In the current study, therefore, we examined the depth-dependent expression
pattern and activity of calpains in the mouse lens. Fluorogenic substrates
were microinjected into the intact lens to visualize calpain activity
directly, and proteomic approaches were used to identify endogenous calpain
substrates. The cleavage pattern of one of these, αII-spectrin, was
examined in detail. Immunocytochemical and immunoblot analysis with wild type
and calpain 3-null lenses indicated that αII-spectrin is a specific
calpain 3 substrate in maturing lens fiber cells. Together, the data suggest
that calpains are activated relatively late in fiber cell differentiation and
may contribute to the remodeling of the membrane cytoskeleton that accompanies
fiber cell maturation. |
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