Feng-Xia Liang

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases.

The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly

Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35–UPIb interaction in the ER is an important early step in urothelial plaque assembly.

Retinoid Signaling in Progenitors Controls Specification and Regeneration of the Urothelium

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

Rab27b is associated with fusiform vesicles and may be involved in targeting uroplakins to urothelial apical membranes

The terminally differentiated umbrella cells of bladder epithelium contain unique cytoplasmic organelles, the fusiform vesicles, which deliver preassembled crystalline arrays of uroplakin proteins to the apical cell surface of urothelial umbrella cells. We have investigated the possible role of Rab proteins in this delivery process, and found Rab27b to be expressed at an extraordinary high level (0.1% of total protein) in urothelium, whereas Rab27b levels were greatly reduced (to <5% of normal urothelium) in cultured urothelial cells, which synthesized only small amounts of uroplakins and failed to form fusiform vesicles. Immuno-electron microscopy showed that Rab27b was associated with the cytoplasmic face of the fusiform vesicles, but not with that of the apical plasma membrane. The association of Rab27b with fusiform vesicles and its differentiation-dependent expression suggest that this Rab protein plays a role in regulating the delivery of fusiform vesicles to the apical plasma membrane of umbrella cells.

Immunomodulation Targeting Abnormal Protein Conformation Reduces Pathology in a Mouse Model of Alzheimer's Disease

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high β-sheet content and resistance to degradation. The most common is Alzheimers disease (AD) where the normal soluble amyloid β (sAβ) peptide is converted into highly toxic oligomeric Aβ and fibrillar Aβ that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAβ and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Aβ or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Aβ in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Aβ40/42 levels, as well as reduced Aβ oligomer levels. This type of immunomodulation has the potential to be a universal β-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases.

Connexin43 and the regulation of intercalated disc function.

Gap junctions mediate the passage of ions and small molecules between cells. In the adult working cardiac ventricles, gap junctions are formed predominantly by oligomerization of the 43 kDa protein connexin43 (Cx43). It is generally accepted that gap junction-mediated intercellular communication is modulated by changes in the intracellular environment. The activity of various kinases, the concentration of protons or calcium, or the association of Cx43 with other intracellular components, all converge to determine the filtering capabilities of intercellular channels. The most studied post-translational modification of Cx43 is the phosphorylation of amino acids in its C-terminal domain (see, e.g1,2). Additional studies have shown that Cx43 is also a substrate for Ne-lysine acetylation.3 Changes in the ionic intracellular milieu can also, directly or indirectly, regulate the conductive state of Cx43. These modulatory mechanisms are likely to be activated in various pathophysiological states.2 Given the key role of gap junctions in action potential propagation, Cx43 regulation is a subject of intense research, and it is seen as an important pharmacological target for the treatment and/or prevention of cardiac arrhythmias.3-6

Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function

We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

Ultrastructure of the intercellular space in adult murine ventricle revealed by quantitative tomographic electron microscopy

AIMS Progress in tissue preservation (high-pressure freezing), data acquisition (tomographic electron microscopy, TEM), and analysis (image segmentation and quantification) have greatly improved the level of information extracted from ultrastructural images. Here, we combined these methods and developed analytical tools to provide an in-depth morphometric description of the intercalated disc (ID) in adult murine ventricle. As a point of comparison, we characterized the ultrastructure of the ID in mice heterozygous-null for the desmosomal gene plakophilin-2 (PKP2; mice dubbed PKP2-Hz). METHODS AND RESULTS Tomographic EM images of thin sections of adult mouse ventricular tissue were processed by image segmentation analysis. Novel morphometric routines allowed us to generate the first quantitative description of the ID intercellular space based on three-dimensional data. We show that complex invaginations of the cell membrane significantly increased the total ID surface area. In addition, PKP2-Hz samples showed increased average intercellular spacing, ID surface area, and membrane tortuosity, as well as reduced number and length of mechanical junctions compared with control. Finally, we observed membranous structures reminiscent of junctional sarcoplasmic reticulum at the ID, which were significantly more abundant in PKP2-Hz hearts. CONCLUSION We have developed a systematic method to characterize the ultrastructure of the intercellular space in the adult murine ventricle and have provided a quantitative description of the structure of the intercellular membranes and of the intercellular space. We further show that PKP2 deficiency associates with ultrastructural defects. The possible importance of the intercellular space in cardiac behaviour is discussed.

MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells

MAL, suggested to play a key role in the apical sorting of membrane proteins, is not involved in the apical sorting of uroplakins. Instead, it plays an important role in facilitating the incorporation of the uroplakin-delivering exocytic vesicles into the apical surface of terminally differentiated urothelial umbrella cells.

Association Between Progranulin and Gaucher Disease.

Background Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of β-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. Methods Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. Findings Serum PGRN levels were significantly lower in GD patients (96.65 ± 53.45 ng/ml) than those in healthy controls of the general population (164.99 ± 43.16 ng/ml, p < 0.0001) and of Ashkenazi Jews (150.64 ± 33.99 ng/ml, p < 0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, “aged” and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. Interpretation Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCases lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.