Christine M. Sorenson

Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair

bcl-2-/-mice complete embryonic development, but display growth retardation and early mortality postnatally. Hematopoiesis including lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution. Thymocytes require an apoptotic signal to manifest accelerated cell death. Renal failure results from severe polycystic kidney disease characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium. bcl-2-/-mice turn gray with the second hair follicle cycle, implicating a defect in redox-regulated melanin synthesis. The abnormalities in these loss of function mice argue that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway.

Tissue Specific Expression of Alternatively Spliced Murine PECAM-1 Isoforms

PECAM‐1 (CD31) is a cell adhesion molecule that is highly expressed at the sites of endothelial cell‐cell contact and at lower levels on the surface of platelets and leukocytes. It is a member of the immunoglobulin gene superfamily and undergoes alternative splicing to generate several isoforms that differ only in their cytoplasmic domains. The tissue distribution of the expression of different PECAM‐1 isoforms has not been previously defined. We have examined PECAM‐1 expression in various mouse tissues and endothelial cells. PECAM‐1 mRNA was highly expressed in lung, heart, and kidney, and to a lower extent in brain and liver. Most endothelial cells in culture expressed high levels of PECAM‐1 mRNA; however, normal mouse brain endothelial cells rapidly lost PECAM‐1 expression in culture. To examine the tissue distribution of PECAM‐1 isoform expression, RT/PCR was performed on the RNA isolated from various mouse tissues and mouse endothelial cells. Cloning and sequencing of the cDNA products indicated that most tissues and endothelial cells expressed several PECAM‐1 isoforms at different frequencies. The PECAM‐1 isoform that lacks exons 14 and 15 was most frequently detected in all cases. A novel PECAM‐1 isoform that lacks exons 12 and 14 was detected in brain. An antibody to the extracellular domain of PECAM‐1 reacted with two major bands, at 130 kDa and 110–120 kDa, in lysates prepared from endothelial cells or kidneys at different stages of development. An antibody prepared against PECAM‐1 exon 14, which reacts only with cytoplasmic domain of PECAM‐1 isoforms that contain exon 14, failed to react with the major lower molecular weight form of PECAM‐1 in these lysates. Therefore, PECAM‐1 isoforms that lack exon 14 are expressed in endothelial cells and tissues in developmentally regulated fashion. These results illustrate that multiple PECAM‐1 isoforms are expressed in various mouse tissues and endothelial cells. Understanding the distribution of PECAM‐1 isoforms, and the identity of intracellular proteins with which they may interact, will help to elucidate the role of PECAM‐1 in endothelial cell–cell interactions and morphogenesis. Dev Dyn 1999;214:44–54.

Focal adhesion kinase, paxillin, and bcl-2: analysis of expression, phosphorylation, and association during morphogenesis.

Cell adhesive mechanisms which determine tissue architecture during morphogenesis are tightly regulated and have an impact on apoptosis, cell migration, proliferation, and differentiation. Bcl‐2 is a death repressor that protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Utilizing the kidney as a model of an organ that undergoes three‐dimensional development we demonstrate that bcl‐2 directly associates with paxillin. Focal adhesion kinase (FAK)(p125) and paxillin(p68) were highly expressed and tyrosine phosphorylated during development but declined to low levels following renal maturation (postnatal day 20) in normal mice. The decline in the expression of p125 FAK and p68 paxillin occurred together with an increase in specific cleavage products of lower molecular weights. Mice deficient in bcl‐2 are born with renal hypoplasia and succumb to renal failure as a result of renal multicystic disease. In kidneys from postnatal day 20 bcl‐2 −/− mice, tyrosine phosphorylation of p125 FAK and p68 paxillin was not down‐regulated. However, the level of expression was similar to that of normal mice. These results demonstrate that the developmentally regulated expression and phosphorylation of FAK and paxillin, in the presence of bcl‐2, is necessary for normal morphogenesis. The interaction of paxillin with bcl‐2 during nephrogenesis may provide an alternative to integrin(s) signaling through paxillin/FAK thus bypassing the need for adhesion‐mediated survival during three dimensional morphogenesis. Dev Dyn 1999;215:371–382.

Nuclear localization of β-catenin and loss of apical brush border actin in cystic tubules of bcl-2 −/− mice

Tight regulation of the rates of cell proliferation and apoptosis is critical for normal nephrogenesis. Nephrogenesis is profoundly affected by the loss of bcl-2 expression. Bcl-2-deficient (bcl-2 -/-) mice are born with renal hypoplasia and succumb to renal failure secondary to renal multicystic disease. Cell-cell and cell-matrix interactions impact tissue architecture by modulating cell proliferation, migration, differentiation, and apoptosis. E-cadherin mediates calcium-dependent homotypic cell-cell interactions that are stabilized by its association with catenins and the actin cytoskeleton. The contribution of altered cell-cell interactions to renal cystic disease has not been delineated. Cystic kidneys from bcl-2 -/- mice displayed nuclear localization of β-catenin and loss of apical brush border actin staining. The protein levels of α-catenin, β-catenin, actin, and E-cadherin were not altered in cystic kidneys compared with normal kidneys. Therefore, an altered distribution of β-catenin and actin, in kidneys from bcl-2 -/- mice, may indicate improper cell-cell interactions interfering with renal maturation and contributing to renal cyst formation.

The human plasminogen activator inhibitor type I gene promoter targets to kidney

The plasminogen activator inhibitor type 1 (PAI-1) gene encodes the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and is induced by cytokines such as transforming growth factor-β (TGF-β). Studies have identified DNA sequence elements within the first 1.3 kb of the 5-upstream DNA that mediate cytokine responsiveness in transfected cells in vitro. However, the DNA sequences that mediate PAI-1 expression in vivo have not yet been delineated. To define these regulatory sequences, we generated transgenic mice that expressed a hybrid gene comprising sequences between -1,272 and +75 of the human PAI-1 gene ligated to a LacZ reporter gene. Transgene expression detected in two independent lines was observed only in kidney from embryonic day 13 to adult and was seen primarily in proximal tubule cells of the outer medulla. Transgene expression and activity were unchanged in response to TGF-β and remained restricted to kidney. Thus we have identified a promoter region within the PAI-1 gene that targets transgene expression to kidney but, unlike the native promoter, is unresponsive to TGF-β in the experimental protocol used.