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Restricted epithelial proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways.

Wang J, Wang N, Xie J, Walton SC, McKown RL, Raab RW, Ma P, Beck SL, Coffman GL, Hussaini IM, Laurie GW - J. Cell Biol. (2006)

Bottom Line: The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1.This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2.Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22904, USA.

ABSTRACT
Renewal of nongermative epithelia is poorly understood. The novel mitogen "lacritin" is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

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Translocated PKCγ becomes associated with PLCα2 and activates PLD1. (A) Perinuclear colocalization of PKCα with PLCγ2; and PKC with PLD1 15 min after lacritin addition. Bars, 25 μm (left) and 10 μm (right). (B) Anti-PLCγ2 immunoprecipitate of homogenate from cells treated for 15 min with lacritin contains PKCα and PLD1. (C) Lacritin dose– response analysis of PLCγ2 phosphorylation in anti-PLCγ2 immunoprecipitates of homogenate from 15-min–treated cells. (D) Lacritin addition promotes activation of PLD in a biphasic dose-dependent manner. Activation is abrogated by knockdown of PKCα using siRNA D7, which has little or no effect on FBS-stimulated PLD activation. Data are presented as the mean ± the SEM.
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fig7: Translocated PKCγ becomes associated with PLCα2 and activates PLD1. (A) Perinuclear colocalization of PKCα with PLCγ2; and PKC with PLD1 15 min after lacritin addition. Bars, 25 μm (left) and 10 μm (right). (B) Anti-PLCγ2 immunoprecipitate of homogenate from cells treated for 15 min with lacritin contains PKCα and PLD1. (C) Lacritin dose– response analysis of PLCγ2 phosphorylation in anti-PLCγ2 immunoprecipitates of homogenate from 15-min–treated cells. (D) Lacritin addition promotes activation of PLD in a biphasic dose-dependent manner. Activation is abrogated by knockdown of PKCα using siRNA D7, which has little or no effect on FBS-stimulated PLD activation. Data are presented as the mean ± the SEM.

Mentions: PKCα regulation of cell proliferation is initiated by translocation of cytoplasmic PKCα to membranes. Interaction of membrane-associated PKCα with other membrane-associated effectors drives downstream signaling to mitogenesis. To appreciate how lacritin mitogenic signaling is transduced in the context of Ca2+ mobilization, we asked whether lacritin promotes PKCα translocation and, if so, to which membrane compartment. PKCα was imaged in cells 15 min after treatment (Fig. 5 A). 10 nM nonmitogenic C-25 had no effect, leaving PKCα diffusely distributed throughout the cytoplasm. In contrast, 10 nM lacritin or N-24 shifted PKCα primarily to the PNG, as confirmed by colocalization with the Golgi marker mannosidase II (Fig. 5 B). Cells treated with positive control PMA concentrated PKCα solely in the PM (unpublished data). The phosphorylation state of PKCα influences its translocation site. Hyperphosphorylated PKCα becomes associated with the PM, whereas hypophosphorylated or dephosphorylated PKCα is known to translocate to the PNG (Hu and Exton, 2004). To determine if the latter is true and, if so, over what time course, cells were treated with lacritin, N-24, and C-25. Cell lysates were then blotted for phospho-PKCα (Fig. 6). 10 nM lacritin promotes PKCα dephosphorylation within 1 min of addition. This form is sustained for at least 15 min, but by 30 min has returned to baseline phosphorylation. Dephosphorylation triggered by increasing molar levels of lacritin or N-24 mirrored the proliferation response, with optimal dephosphorylation at 1 or 10 nM. Nonmitogenic C-25 had no effect, whereas positive control PMA promoted some phosphorylation. It was suggested that lacritin-stimulated signaling toward Ca2+ mobilization follows a Gαi or Gαo–PKCα–PLC pathway. If so, PTX and Go 6976, but not U73122 should inhibit PKCα translocation. We retested each and observed that lacritin-dependent PKCα translocation and dephosphorylation were inhibited by 100 ng/ml PTX and 1 μM Go 6976, but also by 1 μM U73122 (Fig. 5 A). The weakly active analogue U73343 is often used as a negative control for U73122. Cells preincubated with 1 μM U73343 displayed full translocation (Fig. 5 A). This implies that an interdependent complex of PKCα and PLC may be intermediate between Gαi or Gαo activation and PKCα translocation, most logically in the PM. Yet as early as 1 min, PKCα was dephosphorylated, and therefore likely already translocated to the PNG. An alternative possibility is that a PLC isomer is stationed in the PNG and when active serves to capture translocating PKCα. PLCγ2 is concentrated in the PNG in mast cells before and after antigen stimulation (Barker et al., 1998). Is PLCγ2 or another PLC isomer located in the PNG of HSG cells and, if so, does it complex with PKCα? Cells were treated for 15 min with 10 nM lacritin, and then immunostained for PKCα and PLCγ2 (Fig. 7). PLCγ2 displays a discrete perinuclear Golgi localization, which partially overlaps with PKCα (Fig. 7 A), suggesting that the two are in sufficient proximity to form a signaling complex. The existence of a PLCγ2–PKCα complex was, including some activated PLCγ2, confirmed by anti-PLCγ2 immunoprecipitation (Fig. 7, B and C).


Restricted epithelial proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways.

Wang J, Wang N, Xie J, Walton SC, McKown RL, Raab RW, Ma P, Beck SL, Coffman GL, Hussaini IM, Laurie GW - J. Cell Biol. (2006)

Translocated PKCγ becomes associated with PLCα2 and activates PLD1. (A) Perinuclear colocalization of PKCα with PLCγ2; and PKC with PLD1 15 min after lacritin addition. Bars, 25 μm (left) and 10 μm (right). (B) Anti-PLCγ2 immunoprecipitate of homogenate from cells treated for 15 min with lacritin contains PKCα and PLD1. (C) Lacritin dose– response analysis of PLCγ2 phosphorylation in anti-PLCγ2 immunoprecipitates of homogenate from 15-min–treated cells. (D) Lacritin addition promotes activation of PLD in a biphasic dose-dependent manner. Activation is abrogated by knockdown of PKCα using siRNA D7, which has little or no effect on FBS-stimulated PLD activation. Data are presented as the mean ± the SEM.
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Related In: Results  -  Collection

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fig7: Translocated PKCγ becomes associated with PLCα2 and activates PLD1. (A) Perinuclear colocalization of PKCα with PLCγ2; and PKC with PLD1 15 min after lacritin addition. Bars, 25 μm (left) and 10 μm (right). (B) Anti-PLCγ2 immunoprecipitate of homogenate from cells treated for 15 min with lacritin contains PKCα and PLD1. (C) Lacritin dose– response analysis of PLCγ2 phosphorylation in anti-PLCγ2 immunoprecipitates of homogenate from 15-min–treated cells. (D) Lacritin addition promotes activation of PLD in a biphasic dose-dependent manner. Activation is abrogated by knockdown of PKCα using siRNA D7, which has little or no effect on FBS-stimulated PLD activation. Data are presented as the mean ± the SEM.
Mentions: PKCα regulation of cell proliferation is initiated by translocation of cytoplasmic PKCα to membranes. Interaction of membrane-associated PKCα with other membrane-associated effectors drives downstream signaling to mitogenesis. To appreciate how lacritin mitogenic signaling is transduced in the context of Ca2+ mobilization, we asked whether lacritin promotes PKCα translocation and, if so, to which membrane compartment. PKCα was imaged in cells 15 min after treatment (Fig. 5 A). 10 nM nonmitogenic C-25 had no effect, leaving PKCα diffusely distributed throughout the cytoplasm. In contrast, 10 nM lacritin or N-24 shifted PKCα primarily to the PNG, as confirmed by colocalization with the Golgi marker mannosidase II (Fig. 5 B). Cells treated with positive control PMA concentrated PKCα solely in the PM (unpublished data). The phosphorylation state of PKCα influences its translocation site. Hyperphosphorylated PKCα becomes associated with the PM, whereas hypophosphorylated or dephosphorylated PKCα is known to translocate to the PNG (Hu and Exton, 2004). To determine if the latter is true and, if so, over what time course, cells were treated with lacritin, N-24, and C-25. Cell lysates were then blotted for phospho-PKCα (Fig. 6). 10 nM lacritin promotes PKCα dephosphorylation within 1 min of addition. This form is sustained for at least 15 min, but by 30 min has returned to baseline phosphorylation. Dephosphorylation triggered by increasing molar levels of lacritin or N-24 mirrored the proliferation response, with optimal dephosphorylation at 1 or 10 nM. Nonmitogenic C-25 had no effect, whereas positive control PMA promoted some phosphorylation. It was suggested that lacritin-stimulated signaling toward Ca2+ mobilization follows a Gαi or Gαo–PKCα–PLC pathway. If so, PTX and Go 6976, but not U73122 should inhibit PKCα translocation. We retested each and observed that lacritin-dependent PKCα translocation and dephosphorylation were inhibited by 100 ng/ml PTX and 1 μM Go 6976, but also by 1 μM U73122 (Fig. 5 A). The weakly active analogue U73343 is often used as a negative control for U73122. Cells preincubated with 1 μM U73343 displayed full translocation (Fig. 5 A). This implies that an interdependent complex of PKCα and PLC may be intermediate between Gαi or Gαo activation and PKCα translocation, most logically in the PM. Yet as early as 1 min, PKCα was dephosphorylated, and therefore likely already translocated to the PNG. An alternative possibility is that a PLC isomer is stationed in the PNG and when active serves to capture translocating PKCα. PLCγ2 is concentrated in the PNG in mast cells before and after antigen stimulation (Barker et al., 1998). Is PLCγ2 or another PLC isomer located in the PNG of HSG cells and, if so, does it complex with PKCα? Cells were treated for 15 min with 10 nM lacritin, and then immunostained for PKCα and PLCγ2 (Fig. 7). PLCγ2 displays a discrete perinuclear Golgi localization, which partially overlaps with PKCα (Fig. 7 A), suggesting that the two are in sufficient proximity to form a signaling complex. The existence of a PLCγ2–PKCα complex was, including some activated PLCγ2, confirmed by anti-PLCγ2 immunoprecipitation (Fig. 7, B and C).

Bottom Line: The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1.This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2.Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22904, USA.

ABSTRACT
Renewal of nongermative epithelia is poorly understood. The novel mitogen "lacritin" is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

Show MeSH
Related in: MedlinePlus