Discussion
One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. Brady et al. [22] demonstrated, by targeted disruption of the mouse alphaA gene, that this protein was essential for the maintenance of lens transparency, possibly by maintaining the solubility of alphaB, or associated proteins, in the lens. These lenses were also reported to be smaller in equatorial and axial dimensions than age matched wild type lens, which was very similar to that which was observed with the double knockout lens. Targeted disruption of the mouse alphaB gene, however, resulted in lenses similar in size to aged-matched wild type lens with no cataracts reported [23]. This indicates that alphaA may play a greater role in maintaining the transparency of the lens then alphaB. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. This was supported by the morphological observation made in this study of no posterior sutures or fiber cells extending to the posterior capsule of the lens, ectopically staining nucleic acids in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk, gross morphological differences in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. None of these morphological differences have been reported in the single alphaA or alphaB knockout mice. It must be noted, however, that the alphaA/BKO mice also lack the HSPB2 gene product [23] and the contribution of this protein to normal lens morphology and functions should not be overlooked. Future studies should address the possible functions of HSPB2 in normal lens.
The results of the current study support the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. Normal differentiation of lens fiber cells consists of a progression from a simple cuboidal epithelial cell, containing a nucleus and a minimal numbers of organelles, to a stratified layer of elongated fiber-like cells, devoid of nuclei and organelles. Differentiation of epithelial cells occurs in the equatorial/bow region of the lens, where epithelial cells begin to elongate and differentiate into fiber cells of uniform cellular shape, arranged in radial columns of cells extending from the anterior epithelium to the posterior capsule. This process did not appear to have proceeded normally in lenses lacking alphaA and alphaB. The morphological observations presented in this study demonstrate that fiber cells in lenses lacking alphaA and alphaB fail to elongate symmetrically from the bow region and therefore do not establish the typical "onion skin" conformation in which cells extend from the anterior epithelium to the posterior capsule. Additionally, in lenses from 54 wk alphaA/BKO lenses, there was a persistence of cell nuclei in deeper cortical regions, and ectopic cell nuclei were present in large numbers in the anterior central cortex. At 5 wks of age cell nuclei were present, adjacent to the posterior capsule. These morphological observations are consistent with a defect in the normal differentiation pathway of lens epithelial cells into fiber cells.
It is unlikely that these alterations in alphaA/BKO mouse lenses result from increased susceptibility of these lenses to light-induced damage in the absence of the molecular chaperone protection afforded by alphaA and alphaB in normal lenses. With the normal time of eye opening at approximately 14 days after birth, the 5 wk old mice had their eyes open and lenses exposed to light for only about 3 weeks prior to morphological analysis. Moreover, these animals had been exposed to only animal facility fluorescent lighting and were protected from UV light by plastic cages. If lack of protection from light-induced damage was the major factor affecting the changes in these lenses, then the bulk of the damage should have resided along the visual axis, particularly in the central anterior epithelium and subcapsular cortex in the 5 wk lenses, but this was not the case. In these lenses, gross morphological changes were apparent in the equatorial and posterior subcapsular regions. These changes included posterior subcapsular nucleic acid staining, absence of posterior sutures, and small irregularly shaped cells, not arranged in any discernable pattern, in the equatorial/bow region. Systemic stress factors crossing the blood/aqueous barrier might explain some morphological changes at the equatorial region, but this would not explain nucleic acid staining in the posterior subcapsular region. In the 5 wk alphaA/BKO lenses, nucleic acid staining in the posterior subcapsular region is consistent with either anterior epithelial cells migrating aberrantly to the posterior pole, or primary fiber cells failing to fully differentiate by 5 wks of age. These two possibilities could not be differentiated in the methods employed, and were beyond the objectives of the current study. Future studies are being designed to address which of these two processes might explain nucleic acid staining in the posterior subcapsular region. Staining in this region was not observed in older alphaA/BKO lenses, suggesting that this pattern was transient. The fate of the nucleic acid-containing cells in the posterior capsular region of younger lenses is not known at this time, nor is the morphology of earlier stage lenses. Future studies with defined objectives to address the development and progression of morphological changes seen in this study are being designed. The morphological differences in alphaA/BKO lenses, compared to age matched wild type lenses, were consistent with the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. In addition, it was clearly evident that alpha-crystallin is necessary for lens transparency.
The final biological event in a lens epithelial cell's life is the transformation from an epithelial cell into a fiber cell, which occurs at the equatorial/bow region of the lens. The newly forming fiber cell continues to differentiate in the cortex until a mature fiber cell devoid of organelles with suture formation at the ends of the cell is formed. This entire process from epithelial cell to mature fiber cell is defined as lens differentiation. The precise spatial and temporal expression of the crystallin proteins in the developing lens may not be simply a consequence of the differentiation process, but instead may play an important, if not essential, role in the differentiation process itself. The results of the current study support this hypothesis.
The exact in vivo molecular mechanisms, by which alpha-crystallin might influence lens epithelial cell differentiation, and maintenance of lens transparency, remain to be determined. AlphaA and alphaB are members of the shsp family [7]. Previous studies have shown that the alpha-crystallin possesses molecular chaperone activity, binding to partially denatured proteins, both in vitro [5] and probably in vivo [6], to inhibit further denaturation. Although this property may be a major contributor to the maintenance of lens clarity, the early changes in the alphaA/BKO lenses indicate a much broader cellular function for alpha-crystallin. Stress proteins have been shown to be expressed in non-stressed cells during development and differentiation [25]. Hsps were shown to be expressed during the differentiation of mammalian osteoblasts and promelocytic leukemia cells [26]. In addition, hsp expression has been shown to accompany growth arrest in human B lymphocytes [27] and macrophage differentiation of HL 60 cells [28]. During myogenic differentiation, mRNA for alphaB increases in conjunction with the induction of mRNA for myogenin, the earliest known event in myogenesis [29]. The addition of exogenous alpha-crystallin to primary bovine lens epithelium was shown to induce rapid changes in cell shape, leading to the formation of lentoid bodies [21]. These studies strongly suggest that the hsp family of proteins has other functions in addition to protecting proteins and cells during stress.
Alpha-crystallin may play a functional role in the cell nucleus and may have a role in regulating the cell cycle. Several heat shock proteins have been found in cell nuclei in the absence of stress [30], and alpha-crystallin has been shown to interact directly with DNA [18]. AlphaB, expressed in transfected CHO cells, has been shown to ectopically localize to interphase nuclei, suggesting a regulatory role for this protein in the nucleus [19]. A subset of immortalized lens epithelial cells from alphaBKO mice have been shown to hyperproliferate [20] suggesting that alphaB may be important in maintaining genomic stability. In lens epithelium derived from alphaAKO lenses, cell growth rates were reported to be 50% lower compared to wild type [31], suggesting a role for alphaA in regulating the cell cycle. All of these findings raise many questions as to the possible role(s) of alpha-crystallin in the nucleus and in cell cycle regulation during differentiation. In the current study, the observation of a disorganized pattern of nuclei localized to the equatorial bow region of alphaA/BKO lenses, and nucleic acid staining of structures throughout the anterior cortex of 54 wk alphaA/BKO lenses, is consistent with a role for alpha-crystallin in the nucleus.
There is extensive evidence from previous studies demonstrating that alpha-crystallin plays a role in the cytoskeletal organization. Both alphaA and alphaB can bind specifically to actin, both in vitro [13] and in vivo [14]. Actin filament formation has been shown to be necessary for the differentiation of lens epithelial cells [15], however, the significance of alpha-crystallin interaction with actin in differentiation is not known. In the lens, alpha-crystallin also forms a complex with type III intermediate filament proteins and the lens-specific beaded filament proteins CP49 and CP115, which may be critical for proper filament assembly [16]. Beaded filament mRNA levels increase greatly in differentiating lens epithelial cells, and have been suggested as a pan-specific marker for lens fiber cells [17]. It is therefore possible that increased synthesis of alpha-crystallin in epithelial cells early in the differentiation process may have profound effects upon the cytoskeleton, which in turn may profoundly affect cell shape and migration. The lack of cellar organization and uniform cell shape at the equatorial region observed in alphaA/BKO lenses supports this hypothesis. Studies are currently underway to characterize cytoskeletal organization in the alphaA/BKO lens.