33 research outputs found
Vascular and liver homeostasis in juvenile mice require endothelial cyclic AMP-dependent protein kinase A
During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKA(iEC) mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program
Regulation of protein traffic in polarized epithelial cells
The plasma membrane of polarized epithelial
cells is divided into apical and basolateral surfaces with
different compositions. Proteins can be sent directly
from the trans Golgi network (TGN) to either surface, or
can be sent first to one surface and then transcytosed to
the other. The glycosyl phosphatidylinositol anchor is a
signal for apical targeting. Signals in the cytoplasmic
domain containing a B-turn determine basolateral
targeting and retrieval, and are related to other shorting
signals. Transcytosed proteins, such as the polymeric
immunoglobulin receptor (pIgR) are endocytosed from
the basolateral surface and delivered to the apical
recycling compartment underneath the apical surface.
This compartment is a central sorting station, as it
receives material from both surfaces and sorts them to
the correct surface. Delivery to the apical surface from
both the TGN and the apical recycling compartment is
regulated by protein kinase A and protein kinase C, and
endocytosis from the apical surface is also regulated by
kinases. Transcytosis of the pIgR is additionally
regulated by phosphorylation of the pIgR and by ligand
binding to the pIgR. Regulation of traffic in polarized
epithelial cells plays a central role in cellular
homeostasis, response to externa1 signals, and
differentiation
The phospholipid PI (3, 4) P 2 is an apical identity determinant
Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity
Absence of Direct Delivery for Single Transmembrane Apical Proteins or Their “Secretory” Forms in Polarized Hepatic Cells
The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells