Woo-Kuen Lo

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC‐induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF‐α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis.

Different mutations of α3 connexin (Cx46 or Gja8) andα 8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense α8 connexin mutation that resulted in the expression of α8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous α8S50P/+ embryonic lenses, but not in homozygous α8S50P/S50P, α8-/- andα 3-/- α8-/- mutant embryonic lenses. We hypothesized that α8-S50P mutant subunits interacted with wild-typeα 3 or α8, or with both subunits to affect fiber cell formation. We found that the combination of mutant α8-S50P and wild-type α8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of α8-S50P and wild-type α3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.

Knock-in of α3 connexin prevents severe cataracts caused by an α8 point mutation

A G22R point mutation in α8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type α3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in α3 connexin expressed on the locus of the endogenous α8 connexin could modulate the severe cataract caused by the α8-G22R mutation. We found that the α3(-/-) α8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the α3(-/-) α8(G22R/KIα3) lens contained relatively normal inner fibers without lens posterior rupture. The α8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of α3(-/-) α8(G22R/KIα3) lenses, but not in those of α3(-/-) α8(G22R/-) lenses. Thus, we hypothesize that the knocked-in α3 connexin subunits interact with the α8-G22R connexin subunits to form functional gap junction channels and rescue the lens phenotype. Using an electrical coupling assay consisting of paired Xenopus oocytes, we demonstrated that only co-expression of mutant α8-G22R and wild-type α3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by α3 connexin subunits alone. Thus, knocked-in α3 connexin and mutant α8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency.

Microtubule configuration and membranous vesicle transport in elongating fiber cells of the rat lens.

This study examines the microtubule configuration and its close association with the Golgi complex and Golgi-derived membranous vesicles in elongating fiber cells of the rat lens. Since fiber cells elongate tremendously during lens differentiation, we hypothesize that a microtubule-based motor system exists in the elongating fiber cells for transporting important membrane proteins and organelles to the target regions for cell growth. The newly synthesized membrane proteins are known to be transported from the trans-Golgi network in the form of vesicles to the target plasma membrane. By thin-section TEM, we observed a large number of vesicles of various sizes and shapes randomly distributed throughout the cytoplasm of elongating fiber cells. Both Golgi complex and vesicles exhibited characteristic normal structural features seen in other cell types and thus represented real vesicular organelles in the fiber cells. A large number of microtubules were regularly arranged into bundles parallel to the long axis of fiber cells as examined in both longitudinal and cross-section views. Many of these microtubules were closely associated or in intimate contact with the Golgi complex and vesicles in elongating fiber cells. The microtubule polarity assay revealed that microtubules exhibited a unidirectional polarity for the entire length of fiber cells as examined in both anterior and posterior cortical fiber segments. Namely, the minus end of microtubules was towards the anterior lens pole while the plus end was headed towards the posterior pole. This suggests that multiple molecular motors such as kinesin and dynein are needed for carrying the vesicles to both lens poles, since conventional kinesin is known to transport vesicular organelles towards the plus end whereas cytoplasmic dynein carries them towards the minus end of microtubules. By immunoblot analysis, we indeed detected the presence of both kinesin (120 kD) and dynein (70 kD) in homogenate prepared from lens cortical fibers. Moreover, immunogold TEM demonstrated that the aquaporin 0 (formally MIP26) antibody was localized on the membranous vesicles as well as plasma membranes of the cortical fiber cells. This study suggests that a microtubule-based motor system exists in the lens and plays an important role in transporting membrane proteins such as aquaporin 0 in the vesicles during fiber cell differentiation and elongation.

Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens.

PURPOSE Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. METHODS The loss of AQP0 in AQP0(-/-) lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0(-/-) lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. RESULTS Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. CONCLUSIONS This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.

Cardiomyocyte-Restricted Deletion of PPARβ/δ in PPARα-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation

It is well documented that PPARα and PPARβ/δ share overlapping functions in regulating myocardial lipid metabolism. However, previous studies demonstrated that cardiomyocyte-restricted PPARβ/δ deficiency in mice leads to severe cardiac pathological development, whereas global PPARα knockout shows a benign cardiac phenotype. It is unknown whether a PPARα-null background would alter the pathological development in mice with cardiomyocyte-restricted PPARβ/δ deficiency. In the present study, a mouse model with long-term PPARβ/δ deficiency in PPARα-null background showed a comparably reduced cardiac expression of lipid metabolism to those of single PPAR-deficient mouse models. The PPARα-null background did not rescue or aggravate the cardiac pathological development linked to cardiomyocyte-restricted PPARβ/δ deficiency. Moreover, PPARα-null did not alter the phenotypic development in adult mice with the short-term deletion of PPARβ/δ in their hearts, which showed mitochondrial abnormalities, depressed cardiac performance, and cardiac hypertrophy with attenuated expression of key factors in mitochondrial biogenesis and defense. The present study demonstrates that cardiomyocyte-restricted deletion of PPARβ/δ in PPARα-null mice causes impaired mitochondrial biogenesis and defense, but no further depression of fatty acid oxidation. Therefore, PPARβ/δ is essential for maintaining mitochondrial biogenesis and defense in cardiomyocytes independent of PPARα.

In vivo and in vitro observations on permeabi diffusion pathways of tracers in rat and frog lenses

Two tracers [horseradish peroxidase (HRP) and lanthanum]with different properties were injected separately into eyes of the rat and frog to determine the diffusion pathways of tracers in the lens. HRP can diffuse rapidly into lens tissue and can also be washed out, whereas lanthanum has opposite properties. In a short (2–5 min) in vivo intravitreal injection of HRP into the rat eye using an automated micro syringe, tracer reached the entire intercellular space between lens epithelium and fiber cells, and between epithelial cells primarily at the peripheral region; however, it reached the central zone in a longer period (10–20 min) of injection, as examined with light microscope and transmission electron microscope. HRP in epithelial intercellular spaces basal to the tight junctions was washed away upon 5–15 min washing with TC-199, but was retained in the spaces apical to the junctions and between epithelium and fiber cells. Lanthanum studies in both rat and frog lenses showed that within 3–4-hr incubation, tracer was seen in epithelial intercellular spaces only basal to the tight junctions at the central epithelium. At the peripheral zone, however, tracer was visualized in the entire intercellular spaces between epithelial cells, and between epithelium and fiber cells. These studies strongly suggest that both tracers seen in the intercellular space between epithelium and fiber cells are derived from the most equatorial region of the lens, and they may not leak out to the anterior lens surface through tight junctions between epithelial cells in the rat and frog. This leads us to conclude that the equatorial region of the lens is the major site of efflux and influx of substances for the anterior lens region.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

Purpose To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end–capping protein. Methods We investigated F-actin and F-actin–binding protein localization in interdigitations of Tmod1+/+ and Tmod1−/− single mature lens fibers. Results F-actin–rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1−/− mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1−/− mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1−/− mature fibers. Conclusions These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin–actin network stabilized by Tmod1. α-Actinin–crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin–associated proteins required for the formation of paddles between lens fibers.

Connexin 50 Functions as an Adhesive Molecule and Promotes Lens Cell Differentiation

Connexins play essential roles in lens homeostasis and development. Here, we identified a new role for Cx50 that mediates cell-cell adhesion function. Cx50 enhanced the adhesive capability of AQP0. Interestingly, the expression of Cx50 alone promoted cell adhesion at a comparable level to AQP0; however, this cell adhesive function was not observed with other lens connexins, Cx43 and Cx46. Moreover, the adhesive property occurred in both homotypic with Cx50 expressed in both pairing cells and heterotypic with Cx50 in only one pairing cell, and this function appears to be unrelated to its role in forming gap junction channels. Cx50 KO lenses exhibited increased intercellular spaces between lens fiber cells. The second extracellular loop domain (E2) is primarily responsible for this adhesive function. Treatment with a fusion protein containing E2 domain inhibited cell adhesion. Furthermore, disruption of cell adhesion by the E2 domains impaired primary lens cell differentiation. Five critical amino acid residues in the E2 domain primarily are involved in cell adhesive function as well as lens epithelial-fiber differentiation. Together, these results suggest that in addition to forming gap junction channels, Cx50 acts as an adhesive molecule that is critical in maintaining lens fiber integrity and epithelial-fiber differentiation.