Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2.

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.

An induction in hepatic HDL secretion associated with reduced ATPase expression.

Linoleic acid-phospholipids stimulate high-density lipoprotein (HDL) net secretion from liver cells by blocking the endocytic recycling of apoA-I. Experiments were undertaken to determine whether apoA-I accumulation in the cell media is associated with membrane ATPase expression. Treatment of HepG2 cells with dilinoeoylphosphatidylcholine (DLPC) increased apoA-I secretion fourfold. DLPC also significantly reduced cell surface F1-ATPase expression and reduced cellular ATP binding cassette (ABC)A1 and ABCG1 protein levels by approximately 50%. In addition, treatment of HepG2 cells with the ABC transporter inhibitor, glyburide, stimulated the apoA-I secretory effects of both DLPC and clofibrate. Pretreatment of HepG2 cells with compounds that increased ABC transport protein levels (TO901317, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, and resveratrol) blocked the DLPC-induced stimulation in apoA-I net secretion. Furthermore, whereas HepG2 cells normally secrete nascent prebeta-HDL, DLPC treatment promoted secretion of alpha-migrating HDL particles. These data show that an linoleic acid-phospholipid induced stimulation in hepatic HDL secretion is related to the expression and function of membrane ATP metabolizing proteins.

Hepatic high-density lipoprotein secretion regulates the mobilization of cell-surface hepatic lipase.

HDL acts much like heparin to liberate hepatic lipase (HL) from cell surface proteoglycans and stimulate triglyceride clearance. Experiments were undertaken to evaluate the effects of factors that stimulate the secretion of HDL from the liver on the release of HL. Treatment of HepG2 cells with linoleic acid phospholipids (LAPL) (12 muM) promotes a similar increase in the accumulation of both HDL and HL in the cell media. LAPL also induce both apoA-I and HL release from primary human hepatocytes. Dilinoleoylphosphatidylcholine has a greater effect on both apoA-I secretion and HL release than palmitoyllinoleoylphosphatidylcholine. HL released from HepG2 cells is inactive and associated with a large HDL complex containing both apoA-I and apoA-II. Inclusion of the PPARalpha inhibitor, MK-886, or MAPK inhibitor, U0126, completely blocks the LAPL-induced apoA-I and HL accumulation in the media. LAPL-treated cell lysates, however, showed no change in HL protein expression nor HL mRNA. LAPL-induced HL release appears to be a consequence of the displacement ability of newly secreted HDL. Overexpression of pre-pro-apoA-I in HepG2 cells increased HL release, while siRNA inhibition of the apoA-I gene reduced HL in the media. The data show that factors that stimulate HDL secretion in hepatocytes act to also increase the release of HL. This may partly explain why HDL therapeutics often impact plasma triglyceride levels.

Phosphatidylinositol acts through mitogen-activated protein kinase to stimulate hepatic apolipoprotein A-I secretion.

Phosphatidylinositol (PI) has been shown to stimulate reverse cholesterol transport in animal models and to increase plasma apolipoprotein (apo) A-I levels and high-density lipoprotein cholesterol in human subjects. The objective of this study was to determine the molecular mechanism through which PI stimulates apo A-I secretion in hepatic cells. PI (12 mumol/L) significantly stimulates apo A-I secretion from HepG2 cells over 24 hours. The stimulation in apo A-I secretion is completely blocked by phospholipase C inhibitors (D609 and U73122) and the Ras inhibitor sulindac sulfide. Apolipoprotein A-I secretion is augmented with a protein kinase C agonist (dioctanoyl glycerol) and inhibited by a protein kinase C inhibitor (dioleoyl ethylene glycol). The PI-induced apo A-I secretion is unaffected by PI-3-kinase inhibitors but is sensitive to mitogen-activated protein kinase (MAPK) inhibitors. Whereas the p38MAPK inhibitor SB203580 has no effect on PI-induced apo A-I secretion, the MAPK kinase 1/2 inhibitor U0126 and the c-Jun-N-terminal kinase/stress-activated protein kinase inhibitor SP600125 block PI-induced apo A-I secretion. PI also increased extracellular-regulated protein kinase 1 and 2 phosphorylation in HepG2 cells in a time-dependent manner. PI does not appear to stimulate apo A-I gene transcription, as cellular apo A-I messenger RNA levels remained unchanged over the 24-hour incubation. However, PI significantly decreases apo A-I binding and degradation in HepG2 cells. Collectively, the data suggest that PI acts through MAPK pathways to increase plasma apo A-I levels by protecting it from reuptake and degradation.