ISSN 16076729, Doklady Biochemistry and Biophysics, 2013, Vol. 453, pp. 297–299. © Pleiades Publishing, Ltd., 2013. Original Russian Text © V.N. Simirskii, M.K. Duncan, M.A. Paltsev, S.V. Suchkov, 2013, published in Doklady Akademii Nauk, 2013, Vol. 453, No. 3, pp. 342–345.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
β1 Integrin As the Integrating Component in Cell–Cell Cooperation for Maintenance of Lens Transparency V. N. Simirskiia, M. K. Duncanb, Academician M. A. Paltsevc, e, and S. V. Suchkovd, e, f, g Received June 21, 2013
DOI: 10.1134/S1607672913060069
The main function of the lens is to focus images on the retina, which has high requirements for its trans parency. The lens is surrounded by a collagen capsule and consists of a single layer of epithelial cells that pro liferate and differentiate into fibers. Fibers gradually lengthen, move to the center of the lens, and form concentric layers. Lens transparency is ensured both at the molecular level (due to formation of highmolec ularweight complexes consisting of cytoplasmic, cytoskeletal, and plasma membrane proteins) and at the cellular level (by an ordered fiber folding). Disrup tion of the spatial organization of fibers as a result of mutations of the proteins involved in cell–cell con tacts and membrane proteins leads to the loss of trans parency by the lens and the cataract development [1]. The adhesion of the lens cells to the capsule through transmembrane proteins integrins plays an important role in maintaining the tissue organization of the lens [2]. Integrin receptors are composed of one α and one β subunits. β1 integrin is involved in the formation of the vast number of receptors because it can react with the majority of α integrins [3]. In the lens, β1 integrin is expressed in all cells, both in the epithelium and in fibers. In lens fibers, it is located not only on the basal membrane, which con tacts with the capsule, but also on the lateral and apical membranes, on which the extracellular matrix is absent [4]. Earlier, we obtained mice with β1 integrin a
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia b Department of Biological Sciences, University of Delaware, Newark, DE, 19716 USA c Kurchatov Institute Russian Research Center, pl. Kurchatova 1, Moscow, 123182 Russia d Evdokimov Moscow State Medical and Dental University, ul. Delegatskaya 20/1, Moscow, 127473 Russia e European Association for Predictive, Preventive, and Personalized Medicine, Brussels, EU f Association for Research in Vision and Ophthalmology, Rockville, MD, USA g International Society for Eye Research, SF, CA, USA
gene knockout in all lens cells (both in the epithelium and in fibers). In these mice, apoptosis of epithelial lens cells was observed, which ultimately resulted in complete resorption of the lens and microphthalmia after birth [5]. These data suggest that β1 integrin is involved in maintaining the viability of the lens epithe lium; however, the role of β1 integrin in the differenti ation and organization of lens fibers remains obscure. In this study, we investigated the morphological and moleculargenetic consequences of the knockout of the integrin β1 gene in fibers with its expression in the lens epithelium being retained. In the study, we used the transgenic mice FVB/N carrying the Cre recombinase gene under the control of the αА crystallin gene promoter [6] and the hybrid mice C57BL (C57BL6 × 129)F1 carrying insertions of LoxP sites on both sides of exon 3 of the β1 integrin gene (The Jackson Laboratory, Bar Harbor, United States). Total DNA was isolated from tissues using the genotyping reagent kit (Qiagen, The Netherlands). PCR was performed using specific primers and the standard amplification reagent kit (Qiagen). Amplifi cation products were separated by agarose gel electro phoresis in the TrisborateEDTA buffer in the pres ence of ethidium bromide. Histological staining with hematoxylin and eosin was performed on 5 µm thick paraffin sections. Immunofluorescence analysis was performed by the standard techniques [7] on 14 µm thick cryostat sections fixed in acetone–methanol (1 : 1) at –20°С. In the analysis we used rat monoclonal anti bodies to β1 integrin (Chemicon, United States; 1 : 200) and secondary antibodies to rat IgG conjugated to flu orochrome AlexaFluor 568 (Molecular Probes, United States; 1 : 200). The cell nuclei were stained with the DNAspecific dye Draq5 (Biostatus Lim ited, Great Britain; 1 : 3000). Sections were embedded in buffered glycerol (90%, TrisHCl buffer, pH 8.0) containing 0.1% pphenylenediamine. The immunof luorescence reaction was analyzed using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Germany). At an age of 2–3 months, the majority of mice homozygous for the flanked β1 integrin gene and car rying the Cre recombinase gene (βflox/flox/Cre+ geno type) developed cataracts, which differed in the time
297
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SIMIRSKII et al.
(a)
(b)
C C LFs
LFs
(c)
(d) Fig. 1. Lenses of (a, c) knockout mice (βflox/flox/Cre+ genotype) and (b, d) wildtype mice (β +/+/Cre geno type). (a, b) View under a darkfield microscope; (c, d) staining of paraffin sections with hematoxylin and eosin. Designations: LFs—lens fibers, C—lens capsule.
of development and degree of lens opacity. Multiple inbreeding crosses and the breeding of mice with a wellexpressed cataract yielded a subline of mice with a pronounced cataract (Figs. 1a, 1b). The lenses with cataracts contained disorganized fibers and multiple cavities, unlike the normal lens, in which the fibers were tightly packed and formed concentric layers (Figs. 1c, 1d). The most characteristic feature was the
lack of contact of the majority of the fibers with the capsule and the presence of numerous small cavities along the lateral surfaces of the fibers. The efficiency and specificity of the β1 integrin gene knockout in mice was confirmed by PCR analysis of genomic DNA isolated from the lens fibers. Basing on the location of loxP sites, the deletion of exon 3 in the β1 integrin gene could be expected. In addition, αАcrystallin gene promoter, which we have used to control the Cre recombinase expression, does not function in the epithelial cells of the lens and is only activated in differentiated lens fibers [6]. With this in mind, we expected that the β1 integrin gene knockout will be limited to the lens fibers. Indeed, PCR analysis of the genomic DNA isolated from the lens fibers of mice with the βflox/flox/Cre+ genotype confirmed the deletion of exon 3 in the β1 integrin gene. It should be noted that the size of the amplification products for the fibers of knockout and wildtype mice differed by the value corresponding to the estimated size of the DNA region between the loxP sites (exon 3 with sur rounding intron regions) (Fig. 2). Corresponding analysis of DNA from the lens epithelium of the mice showed that exon 3 in the knockout mice was not deleted. β1 Integrin was immunochemically detected in the epithelium; in the fibers, it was localized only in the superficial layers in the equatorial region of the lens at all stages of embryonic development. In the deeper layers of the fibers, β1 integrin was not detected starting from 15.5–16.5 days post conception (dpc) (Fig. 3). This agrees with the start of differentiation of secondary fibers of the lens and subsequent activation of the Cre recombinase on 13.5 dpc [6, 8]. The delay in the pro tein loss was apparently due to the fact that β1 integrin synthesized in the epithelium is not degraded immedi ately and is stored for some time in the early fibers dif ferentiated from the epithelial cells. The preferential localization of β1 integrin in the epithelium (with its
(a)
F −
(b) bp loxP
Before knockout Exon 3 F − After knockout
935
loxP − R
Exon 4 390
loxP − R
Exon 4
1
2
3
Fig. 2. PCR analysis of genomic DNA from mouse lens fibers. (a) Scheme of the β1 integrin gene region with the loxP sites before and after knockout. The positions of primers that were used for PCR analysis are indicated. (b) Electrophoretogram of PCR prod ucts. Designations: 1—wildtype allele (β+/+/Cre+), 2—heterozygote (βflox/+/Cre+), 3—homozygote (βflox/flox/Cre+), loxP—loxP site, F—forward primer, R—reverse primer; bp—base pairs. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 453
2013
β1 INTEGRIN AS THE INTEGRATING COMPONENT
the cell–cell contact proteins (nectins and connexins) in fibroblasts cultured in vitro [11]. Thus, the β1 integrin gene knockout in the lens fibers leads to the disturbance of their spatial organiza tion, which is caused, on the one hand, by the loss of contact with the basal part of the lens capsule fibers, on the other hand, by disturbed cellcell communica tion between the lateral surfaces of the fibers. These data suggest that β1 integrin, in addition to its primary function (providing adhesion to the extracellular matrix) also functions as a peculiar integrating link between the proteins of cell–cell contacts and the cytoskeleton, which promotes proper packaging of the fibers and is essential for the maintenance of lens transparency.
LE LFs
(a)
(b)
LE
LFs
(c)
(d) Fig. 3. Immunofluorescent localization of β1 integrin on cryostat sections of the lens of 16.5 days mouse embryos: (a) section of the lens of a wildtype mouse stained with antibodies to β1 integrin; (b) the same section stained for DNA; (c) section of the lens of a knockout mice stained with antibodies to β1 integrin; (d) the same section stained for DNA. Designations: LFs—lens fibers, LE—lens epi thelium. Scale, 77 µm.
absence in the majority of the fibers) was retained in the lens of adult knockout mice irrespective of the degree of the cataract development. Lens fibers are highly elongated cells that tightly adjoin one another through their lateral surfaces. In a normal lens, the fibers of the surface and middle layers are attached by their basal surfaces to the capsule through the adhesive contacts containing β1 integrin [9]. In addition, the tight adhesion of the fibers to one another is reached by interdigitizing protrusions on their lateral surfaces [10]. These areas also contain β1 integrin but lack extracellular matrix. Apparently, in this case β1 integrin can function as an additional integrating factor, providing the interaction of proteins of cell–cell contacts with the actin filament system within the fibers. This possibility was demonstrated, in particular, for αVβ3 integrins, which can interact with
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ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project no. 120400186a), the program of the Presidium of the Russian Academy of Sciences “Wildlife: Current Status and Problems of Development,” and the US National Eye Institute (grant no. EY015279). REFERENCES 1. Churchill, A. and Graw, J., Phil. Trans. R. Soc. B, 2011, vol. 366, pp. 1234–1249. 2. Walker, J. and Menko, A.S., Exp. Eye Res., 2009, vol. 88, pp. 216–225. 3. Hynes, R.O., Cell, 2002, vol. 110, pp. 673–687. 4. Menko, A.S. and Philip, N.J., Exp. Cell Res., 1995, vol. 218, pp. 516–521. 5. Simirskii, V.N., Wang, Y., and Duncan, M.K., Dev. Biol., 2007, vol. 306, pp. 658–668. 6. Zhao, H., Yang, Y., Rizo, C.M., et al., Invest. Ophthal mol. Visual Sci., 2004, vol. 45, pp. 1930–1939. 7. Reed, N.A., Oh, D.J., Czymmek, K.J., and Dun can, M.K., J. Immunol. Met., 2001, vol. 253, pp. 243– 252. 8. Lovicu, F.J. and McAvoy, J.W., Dev. Biol., 2005, vol. 280, pp. 1–14. 9. Lu, J.Y., Mohammed, T.A., Donohue, S.T., et al., Mol. Vis., 2008, vol. 14, pp. 1187–1203. 10. Biswas, S.K., Lee, J.E., Brako, L., et al., Mol. Vis., 2010, vol. 16, pp. 2328–2341. 11. Sakamoto, T., Ogita, H., Hirota, T., et al., J. Biol. Chem., 2006, vol. 281, pp. 19631–19644.
Translated by M. Batrukova
2013
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
β1 Integrin As the Integrating Component in Cell–Cell Cooperation for Maintenance of Lens Transparency V. N. Simirskiia, M. K. Duncanb, Academician M. A. Paltsevc, e, and S. V. Suchkovd, e, f, g Received June 21, 2013
DOI: 10.1134/S1607672913060069
The main function of the lens is to focus images on the retina, which has high requirements for its trans parency. The lens is surrounded by a collagen capsule and consists of a single layer of epithelial cells that pro liferate and differentiate into fibers. Fibers gradually lengthen, move to the center of the lens, and form concentric layers. Lens transparency is ensured both at the molecular level (due to formation of highmolec ularweight complexes consisting of cytoplasmic, cytoskeletal, and plasma membrane proteins) and at the cellular level (by an ordered fiber folding). Disrup tion of the spatial organization of fibers as a result of mutations of the proteins involved in cell–cell con tacts and membrane proteins leads to the loss of trans parency by the lens and the cataract development [1]. The adhesion of the lens cells to the capsule through transmembrane proteins integrins plays an important role in maintaining the tissue organization of the lens [2]. Integrin receptors are composed of one α and one β subunits. β1 integrin is involved in the formation of the vast number of receptors because it can react with the majority of α integrins [3]. In the lens, β1 integrin is expressed in all cells, both in the epithelium and in fibers. In lens fibers, it is located not only on the basal membrane, which con tacts with the capsule, but also on the lateral and apical membranes, on which the extracellular matrix is absent [4]. Earlier, we obtained mice with β1 integrin a
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia b Department of Biological Sciences, University of Delaware, Newark, DE, 19716 USA c Kurchatov Institute Russian Research Center, pl. Kurchatova 1, Moscow, 123182 Russia d Evdokimov Moscow State Medical and Dental University, ul. Delegatskaya 20/1, Moscow, 127473 Russia e European Association for Predictive, Preventive, and Personalized Medicine, Brussels, EU f Association for Research in Vision and Ophthalmology, Rockville, MD, USA g International Society for Eye Research, SF, CA, USA
gene knockout in all lens cells (both in the epithelium and in fibers). In these mice, apoptosis of epithelial lens cells was observed, which ultimately resulted in complete resorption of the lens and microphthalmia after birth [5]. These data suggest that β1 integrin is involved in maintaining the viability of the lens epithe lium; however, the role of β1 integrin in the differenti ation and organization of lens fibers remains obscure. In this study, we investigated the morphological and moleculargenetic consequences of the knockout of the integrin β1 gene in fibers with its expression in the lens epithelium being retained. In the study, we used the transgenic mice FVB/N carrying the Cre recombinase gene under the control of the αА crystallin gene promoter [6] and the hybrid mice C57BL (C57BL6 × 129)F1 carrying insertions of LoxP sites on both sides of exon 3 of the β1 integrin gene (The Jackson Laboratory, Bar Harbor, United States). Total DNA was isolated from tissues using the genotyping reagent kit (Qiagen, The Netherlands). PCR was performed using specific primers and the standard amplification reagent kit (Qiagen). Amplifi cation products were separated by agarose gel electro phoresis in the TrisborateEDTA buffer in the pres ence of ethidium bromide. Histological staining with hematoxylin and eosin was performed on 5 µm thick paraffin sections. Immunofluorescence analysis was performed by the standard techniques [7] on 14 µm thick cryostat sections fixed in acetone–methanol (1 : 1) at –20°С. In the analysis we used rat monoclonal anti bodies to β1 integrin (Chemicon, United States; 1 : 200) and secondary antibodies to rat IgG conjugated to flu orochrome AlexaFluor 568 (Molecular Probes, United States; 1 : 200). The cell nuclei were stained with the DNAspecific dye Draq5 (Biostatus Lim ited, Great Britain; 1 : 3000). Sections were embedded in buffered glycerol (90%, TrisHCl buffer, pH 8.0) containing 0.1% pphenylenediamine. The immunof luorescence reaction was analyzed using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Germany). At an age of 2–3 months, the majority of mice homozygous for the flanked β1 integrin gene and car rying the Cre recombinase gene (βflox/flox/Cre+ geno type) developed cataracts, which differed in the time
297
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SIMIRSKII et al.
(a)
(b)
C C LFs
LFs
(c)
(d) Fig. 1. Lenses of (a, c) knockout mice (βflox/flox/Cre+ genotype) and (b, d) wildtype mice (β +/+/Cre geno type). (a, b) View under a darkfield microscope; (c, d) staining of paraffin sections with hematoxylin and eosin. Designations: LFs—lens fibers, C—lens capsule.
of development and degree of lens opacity. Multiple inbreeding crosses and the breeding of mice with a wellexpressed cataract yielded a subline of mice with a pronounced cataract (Figs. 1a, 1b). The lenses with cataracts contained disorganized fibers and multiple cavities, unlike the normal lens, in which the fibers were tightly packed and formed concentric layers (Figs. 1c, 1d). The most characteristic feature was the
lack of contact of the majority of the fibers with the capsule and the presence of numerous small cavities along the lateral surfaces of the fibers. The efficiency and specificity of the β1 integrin gene knockout in mice was confirmed by PCR analysis of genomic DNA isolated from the lens fibers. Basing on the location of loxP sites, the deletion of exon 3 in the β1 integrin gene could be expected. In addition, αАcrystallin gene promoter, which we have used to control the Cre recombinase expression, does not function in the epithelial cells of the lens and is only activated in differentiated lens fibers [6]. With this in mind, we expected that the β1 integrin gene knockout will be limited to the lens fibers. Indeed, PCR analysis of the genomic DNA isolated from the lens fibers of mice with the βflox/flox/Cre+ genotype confirmed the deletion of exon 3 in the β1 integrin gene. It should be noted that the size of the amplification products for the fibers of knockout and wildtype mice differed by the value corresponding to the estimated size of the DNA region between the loxP sites (exon 3 with sur rounding intron regions) (Fig. 2). Corresponding analysis of DNA from the lens epithelium of the mice showed that exon 3 in the knockout mice was not deleted. β1 Integrin was immunochemically detected in the epithelium; in the fibers, it was localized only in the superficial layers in the equatorial region of the lens at all stages of embryonic development. In the deeper layers of the fibers, β1 integrin was not detected starting from 15.5–16.5 days post conception (dpc) (Fig. 3). This agrees with the start of differentiation of secondary fibers of the lens and subsequent activation of the Cre recombinase on 13.5 dpc [6, 8]. The delay in the pro tein loss was apparently due to the fact that β1 integrin synthesized in the epithelium is not degraded immedi ately and is stored for some time in the early fibers dif ferentiated from the epithelial cells. The preferential localization of β1 integrin in the epithelium (with its
(a)
F −
(b) bp loxP
Before knockout Exon 3 F − After knockout
935
loxP − R
Exon 4 390
loxP − R
Exon 4
1
2
3
Fig. 2. PCR analysis of genomic DNA from mouse lens fibers. (a) Scheme of the β1 integrin gene region with the loxP sites before and after knockout. The positions of primers that were used for PCR analysis are indicated. (b) Electrophoretogram of PCR prod ucts. Designations: 1—wildtype allele (β+/+/Cre+), 2—heterozygote (βflox/+/Cre+), 3—homozygote (βflox/flox/Cre+), loxP—loxP site, F—forward primer, R—reverse primer; bp—base pairs. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 453
2013
β1 INTEGRIN AS THE INTEGRATING COMPONENT
the cell–cell contact proteins (nectins and connexins) in fibroblasts cultured in vitro [11]. Thus, the β1 integrin gene knockout in the lens fibers leads to the disturbance of their spatial organiza tion, which is caused, on the one hand, by the loss of contact with the basal part of the lens capsule fibers, on the other hand, by disturbed cellcell communica tion between the lateral surfaces of the fibers. These data suggest that β1 integrin, in addition to its primary function (providing adhesion to the extracellular matrix) also functions as a peculiar integrating link between the proteins of cell–cell contacts and the cytoskeleton, which promotes proper packaging of the fibers and is essential for the maintenance of lens transparency.
LE LFs
(a)
(b)
LE
LFs
(c)
(d) Fig. 3. Immunofluorescent localization of β1 integrin on cryostat sections of the lens of 16.5 days mouse embryos: (a) section of the lens of a wildtype mouse stained with antibodies to β1 integrin; (b) the same section stained for DNA; (c) section of the lens of a knockout mice stained with antibodies to β1 integrin; (d) the same section stained for DNA. Designations: LFs—lens fibers, LE—lens epi thelium. Scale, 77 µm.
absence in the majority of the fibers) was retained in the lens of adult knockout mice irrespective of the degree of the cataract development. Lens fibers are highly elongated cells that tightly adjoin one another through their lateral surfaces. In a normal lens, the fibers of the surface and middle layers are attached by their basal surfaces to the capsule through the adhesive contacts containing β1 integrin [9]. In addition, the tight adhesion of the fibers to one another is reached by interdigitizing protrusions on their lateral surfaces [10]. These areas also contain β1 integrin but lack extracellular matrix. Apparently, in this case β1 integrin can function as an additional integrating factor, providing the interaction of proteins of cell–cell contacts with the actin filament system within the fibers. This possibility was demonstrated, in particular, for αVβ3 integrins, which can interact with
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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Vol. 453
ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project no. 120400186a), the program of the Presidium of the Russian Academy of Sciences “Wildlife: Current Status and Problems of Development,” and the US National Eye Institute (grant no. EY015279). REFERENCES 1. Churchill, A. and Graw, J., Phil. Trans. R. Soc. B, 2011, vol. 366, pp. 1234–1249. 2. Walker, J. and Menko, A.S., Exp. Eye Res., 2009, vol. 88, pp. 216–225. 3. Hynes, R.O., Cell, 2002, vol. 110, pp. 673–687. 4. Menko, A.S. and Philip, N.J., Exp. Cell Res., 1995, vol. 218, pp. 516–521. 5. Simirskii, V.N., Wang, Y., and Duncan, M.K., Dev. Biol., 2007, vol. 306, pp. 658–668. 6. Zhao, H., Yang, Y., Rizo, C.M., et al., Invest. Ophthal mol. Visual Sci., 2004, vol. 45, pp. 1930–1939. 7. Reed, N.A., Oh, D.J., Czymmek, K.J., and Dun can, M.K., J. Immunol. Met., 2001, vol. 253, pp. 243– 252. 8. Lovicu, F.J. and McAvoy, J.W., Dev. Biol., 2005, vol. 280, pp. 1–14. 9. Lu, J.Y., Mohammed, T.A., Donohue, S.T., et al., Mol. Vis., 2008, vol. 14, pp. 1187–1203. 10. Biswas, S.K., Lee, J.E., Brako, L., et al., Mol. Vis., 2010, vol. 16, pp. 2328–2341. 11. Sakamoto, T., Ogita, H., Hirota, T., et al., J. Biol. Chem., 2006, vol. 281, pp. 19631–19644.
Translated by M. Batrukova
2013
