Structural function of MIP/aquaporin 0 in the eye lens; genetic defects lead to congenital inherited cataracts.

Aquaporin 0 (AQP0) was originally characterized as a membrane intrinsic protein, specifically expressed in the lens fibers of the ocular lens and designated MIP, for major intrinsic protein of the lens. Once the gene was cloned, an internal repeat was identified, encoding for the amino acids Asp-Pro-Ala, the NPA repeat. Shortly, the MIP gene family was emerging, with members being characterized in mammals, insects, and plants.

PKC putative phosphorylation site Ser235 is required for MIP/AQP0 translocation to the plasma membrane.

Purpose: To investigate the functional significance of MIP/AQP0 phosphorylation at serine(235).

Methods: MIP/AQP0 expression and cellular localization was studied in rat lens epithelia explants induced to differentiate by FGF-2. MIP wild type (WT) and MIP (S235A) mutant expression plasmids were constructed and transiently expressed in RK13 cells.

Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis.

Specific interaction between lens MIP/Aquaporin-0 and two members of the gamma-crystallin family.

Purpose: Major Intrinsic Protein (MIP)/Aquaporin 0 is required for lens transparency and is specifically expressed in lens fiber cell membranes. We have demonstrated previously that in the rat lens MIP interacts specifically with gammaE-crystallin, resulting in its recruitment to the plasma membrane. Our goal was to examine the interaction or lack of interaction between MIP and all members of the gamma-crystallin family and to provide evidence for a physiological role these interactions may play in gamma-crystallin or MIP function.

Lens major intrinsic protein (MIP)/aquaporin 0 expression in rat lens epithelia explants requires fibroblast growth factor-induced ERK and JNK signaling.

Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2.

Activation of STAT signaling pathways and induction of suppressors of cytokine signaling (SOCS) proteins in mammalian lens by growth factors.

Purpose: This study was conducted to examine whether the effects of growth factors are mediated in the lens by Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways and whether they induce expression of suppressors of cytokine signaling (SOCS), a novel family of feedback regulators of cytokine and growth factor activities.

Methods: STAT activation and SOCS expression were analyzed in transgenic or wild-type mouse lens and lens epithelial cells stimulated with growth factors by immunohistochemistry, RT-PCR, Northern, Western, proliferation, or transient reporter assays.

Results: STATs were constitutively expressed at low levels and activated by insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-aa, and FGF-1 or -2 in the lens.

gammaE-crystallin recruitment to the plasma membrane by specific interaction between lens MIP/aquaporin-0 and gammaE-crystallin.

Purpose: Major intrinsic protein (MIP), also called aquaporin-0, is essential for lens transparency and is specifically expressed in the lens fiber cell membranes. The goal of the current study was to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions.

Methods: The C-terminal 74-amino-acid fragment of MIP was used as bait to screen a rat lens cDNA yeast two-hybrid library.

Characterization of the roles of STAT1 and STAT3 signal transduction pathways in mammalian lens development.

Purpose: IGF-1 and PDGF are implicated in regulating lens proliferation and/or providing spatial cues that restrict lens proliferation to germinative and transition zones of the lens. However, very little is known about how IGF-1- or PDGF-induced signals are transduced and coupled to gene transcription in lens cells. Therefore, we examined whether these growth factors mediate their effects in the lens through the evolutionarily conserved JAK/STAT signal transduction pathway and if STAT signaling is essential for mammalian lens development.

Mafs, Prox1, and Pax6 can regulate chicken betaB1-crystallin gene expression.

During lens fiber cell differentiation, the regulation of crystallin gene expression is coupled with dramatic morphological changes. Here we report that Mafs, Prox1, and Pax6, which are essential transcription factors for normal lens development, bind to three functionally important cis elements, PL1, PL2, and OL2, in the chicken betaB1-crystallin promoter and may cooperatively direct the transcription of this lens fiber cell preferred gene. Gel shift assays demonstrated that Mafs bind to the MARE-like sequences in the PL1 and PL2 elements, whereas Prox1, a sequence-specific DNA-binding protein like its Drosophila homolog Prospero, interacts with the OL2 element.

pH-Dependent channel activity of heterologously-expressed main intrinsic protein (MIP) from rat lens.

Wild-type rat lens main intrinsic protein (MIP) was heterologously expressed in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and in mouse erythroid leukaemia cells (MEL C88). Both MEL and Sf21 cell lines expressing wild-type MIP were investigated for the conductance of ions using a whole cell patch clamp technique. An increase in conductance was seen in both expression systems, particularly on lowering the pH to 6.

Heterologous expression and topography of the main intrinsic protein (MIP) from rat lens.

Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography.