at2-specific knockout mice  (Jackson Laboratory)

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency results in AT2 hypertrophy and changes to immune response and stress pathways. A , immunohistochemical staining on lung sections indicate Mfsd2a ( red ) expression in AT2 cells of 2a fl/fl mice but absent in AT2aKO. Pdpn ( green ) stains AT1 cells. 2a fl/fl , n = 6; AT2aKO, n = 4. The scale bar represents 50 μm. Inset in images is the enlarged area indicated by white box . The scale bar represents 12.5 μm. B , H&E-stained lung section of AT2aKO and 2a fl/fl control 4 weeks post-tamoxifen. n = 6 per genotype. Masson's trichome-stained lung section of AT2aKO and 2a fl/fl control 12 weeks post-tamoxifen. 2a fl/fl , n = 3; AT2aKO, n = 7. AT2 cells are indicated by black arrows . The scale bar represents 20 μm. Inset in images is the enlarged area indicated by black box . The scale bar represents 10 μm. C , quantification of AT2 cell diameter from AT2aKO and 2a fl/fl controls at 4 and 12 weeks post-tamoxifen represented as a scatterplot. Unpaired t test with Welch's correction, ∗∗∗∗ p < 0.0001. Biological replicates as indicated in B . D , RNA-Seq analysis of AT2aKO and 2a fl/fl AT2 cells. Bubble plot represents top 20 significantly upregulated or downregulated Gene Ontology terms. E , heatmap highlighting inflammatory and fibrosis markers that were upregulated in AT2aKO relative to 2a fl/fl controls. Color bar indicates z -score transformation on median ratio normalized counts. n = 4 per genotype. Wald test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. AT1, alveolar epithelial type 1; AT2, alveolar epithelial type 2; Mfsd2a, major facilitator superfamily domain containing 2a.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Immunohistochemical staining, Staining, Expressing, Control, RNA Sequencing, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a mediates the uptake of LPCs into AT2 at the apical surface. A , orthoview of z-stack of lung sections stained for Mfsd2a ( red ) and the AT2 basolateral membrane marker Na + /K + ATPase α1 ( green ). The scale bar represents 20 μm. B , 3D reconstruction of confocal images in ( A ) indicated enrichment of Mfsd2a ( red ) at the apical side of AT2 cells. 2a fl/fl , n = 6; AT2aKO, n = 4. The scale bar represents 50 μm. C , intratracheal instillation of LPC-NBD. Immunohistochemical staining of Sftpc ( red ) and F4/80 ( gray ) on lung sections indicates accumulation of LPC-NBD ( green ) only in 2a fl/fl AT2 cells but absent in AT2aKO AT2. LPC-NBD accumulation in macrophages is observed in both AT2aKO and 2a fl/fl AT2. The scale bar represents 20 μm. Inset in images is the enlarged area indicated by white box . The scale bar represents 10 μm. n = 4 per genotype. D , intravenous instillation of LPC-NBD. No LPC-NBD uptake was observed in AT2aKO or 2a fl/fl AT2 cells. Sftpc ( red ) stains AT2 cells. n = 4 per genotype. The scale bar represents 50 μm. E , LPC-NBD ( green ) was taken up by AT2 cells isolated from 2a fl/fl but not AT2aKO mice in vitro . Sftpc ( red ) denotes AT2 cells. Hoechst ( blue ) denotes nuclei. n = 5 per genotype. The scale bar represents 20 μm. AT2, alveolar epithelial type 2; LPC, lysophosphatidylcholine; Mfsd2a, major facilitator superfamily domain containing 2a; NBD, [12-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-dodecanoyl.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Staining, Membrane, Marker, Immunohistochemical staining, Isolation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Uptake of LPC by AT2 cells is dependent on lung phospholipase A 2 (PLA 2 ) activity. A , intratracheal instillation of Red/Green BODIPY PC-A2. Both red LPC and green FA are taken up by 2a fl/fl AT2 as seen by colocalization with Sftpc (indicated by white arrows in insets of area denoted by white squares ), whereas only green FA is taken up by AT2aKO AT2. B , red LPC is also taken up by macrophages as seen by colocalization with F4/80 (indicated by white arrows in insets of area denoted by white squares ) in both AT2aKO and 2a fl/fl AT2. C , quantification of red and green fluorescence in lung sections. 2a fl/fl , n = 5; AT2aKO, n = 4. The scale bar represents 50 μm; the scale bar ( inset ) represents 12.5 μm. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01. AT2, alveolar epithelial type 2; FA, fatty acid; LPC, lysophosphatidylcholine.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Assessment of sPLA 2 activity in BAL. sPLA 2 activity was measured in BAL isolated from AT2aKO or 2a fl/fl using Red/Green BODIPY PC ( A and B ) or Bis-BODIPY FL C11-PC ( C ), and lipids were extracted and resolved by TLC. sPLA 2 activity was comparable between both genotypes. LPC and FA were indicated by red and blue arrows , respectively. Unhydrolyzed PC substrates (no BAL, black arrows ) are fluorescent in organic solvent used for TLC analysis as these substrates are no longer ordered and quenched as they are in BAL in vivo . 2a fl/fl , n = 5; AT2aKO, n = 3. D , quantification of sPLA 2 activity is represented as the mean ± SE of the ratio of the sum of products (LPC and FA) over the sum of products and unhydrolyzed substrate. 2a fl/fl , n = 5; AT2aKO, n = 3. BAL, bronchioalveolar lavage; FA, fatty acid; FL, full-length; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; sPLA 2 , secretory PLA 2 .

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay, Isolation, Solvent, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency leads to reductions in total BAL phospholipids (PLs). Lipidomic analysis of AT2aKO and 2a fl/fl BAL 4 weeks post-tamoxifen treatment. A , BAL lipid species represented as a volcano plot. Dots above dashed horizontal line indicate lipid species that are significantly different between AT2aKO and 2a fl/fl AT2. Most of the lyso-PL and PL species are reduced in AT2aKO BAL relative to 2a fl/fl control (PL, SL, and NL). Total PC 32:0 ( B ) and PL species containing DHA (PL-DHA) ( C ) or AA (PL-AA) ( D ) were reduced in AT2aKO BAL relative to 2a fl/fl . BAL PLs ( E ) and lyso-PLs ( F ) represented as a heatmap. Color bar indicates z -score transformation on μmol/μl BAL PLs. Data for B – D are represented as mean μmol/μl BAL ± SE, with individual points denoting biological replicates. Fatty acid identity is designated as the number of carbons:number of double bonds. For all panels in this figure, 2a fl/fl , n = 6; AT2aKO, n = 5. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. AA, arachidonic acid; AT2, alveolar epithelial type 2; BAL, bronchioalveolar lavage; DHA, docosahexaenoic acid; Mfsd2a, major facilitator superfamily domain containing 2a; NL, neutral lipid; PC, phosphatidylcholine; SL, sphingolipid.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Control, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency alters the AT2 cell lipidome reflective of changes in BAL. Lipidomic analysis of AT2aKO and 2a fl/fl BAL 2 weeks post-tamoxifen treatment. Total PC 32:0 ( A ) and phospholipid (PL) species containing DHA (PL-DHA) ( B ) or AA (PL-AA) ( C ) were reduced in AT2aKO BAL relative to 2a fl/fl . D , lipidomic analysis of AT2aKO and 2a fl/fl AT2 cells 2 weeks post-tamoxifen treatment. Total PC 32:0 was reduced in AT2aKO AT2 cells relative to 2a fl/fl . E , mol% lipid species represented as a volcano plot. Dots above dashed line indicate lipid species that are significantly different between AT2aKO and 2a fl/fl AT2 (PL, SL, and NL. F , PLs with monounsaturated fatty acids (PL-MUFA) species were increased in AT2aKO AT2 cells relative to 2a fl/fl . G , PC–DHA species were reduced in AT2aKO AT2 cells relative to 2a fl/fl , similar to changes in the BAL lipidome. For A – D , data are represented as mean μmol/μl BAL ± SE, with individual points denoting biological replicates. 2a fl/fl , n = 13; AT2aKO, n = 12. For F – H , data represented as mol% ± SE of PLs, with individual points denoting biological replicates. n = 7 per genotype. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. H , model to show Mfsd2a is required for surfactant homeostasis. AT2 cells synthesize surfactants, which are secreted as DPPC-rich lamellar bodies at the apical surface that unravel into large surfactant aggregates that make up the alveolar lining fluid. Spent surfactant is released as small surfactant aggregates that are hydrolyzed by sPLA 2 in the alveolar space to produce lysophosphatidylcholines (LPCs). LPC are then taken up by Mfsd2a expressed at the apical surface of AT2 cells and serve as a precursor for the synthesis of PC–DHA that are then remodeled into the main surfactant PL DPPC. Some LPCs can also be cleared by alveolar macrophages. In the absence of Mfsd2a, uptake of LPCs is limited resulting in reduction in PC–DHA leading to reduced resynthesis of DPPC. Moreover, reduced sPLA 2 activity in the absence of Mfsd2a might be a result of altered surfactant composition.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay

Journal:

Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

doi: 10.1016/j.cellsig.2010.07.012

Figure Lengend Snippet: AT1 (A) and AT2 (B) were detected in kidney slices as described in the methods section. Arrows indicate localization of AT1 and AT2. Magnification = 20X. AT1 is strongly expressed in the apical and lateral membranes of the tubules (thin arrows), and in mesangial cells (thick arrow),.AT2 is expressed mostly in glomerular endothelial cells (thick arrow) and in the proximal tubules (thin arrow). The pictures are representative of kidneys from 2 individual mice. C) detection of AT1 and AT2 by western blot in kidney cortex homogenates using the same antibodies as in A and B.

Article Snippet: Age- and sex-matched AT2 knockout mice (a generous gift from Dr Victor Dzau, Duke University) and their wild-type controls (129S6/SvEvTac, Jackson Laboratories, Bar Harbor, ME) received STZ as described above and were sacrificed 24 hours after onset of hyperglycemia.

Techniques: Western Blot

Journal:

Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

doi: 10.1016/j.cellsig.2010.07.012

Figure Lengend Snippet: A. Angiotensin (Ang I and Ang II) concentration in kidney cortex homogenates was determined by ELISA. **p<0.01 by Student's t-test. Activation of AT1 (B) and AT2 (C) was assessed on kidney cortex homogenates by immunoblot using conformation-specific antibodies. For each analysis, loading control was assessed by detection of the corresponding receptor, regardless of its activation state, using antibodies directed against the extracellular part of the receptors. The lower panel shows combined data obtained on kidneys from 5 individual mice in each group. **p<0.01, ns = not significant by ANOVA.

Article Snippet: Age- and sex-matched AT2 knockout mice (a generous gift from Dr Victor Dzau, Duke University) and their wild-type controls (129S6/SvEvTac, Jackson Laboratories, Bar Harbor, ME) received STZ as described above and were sacrificed 24 hours after onset of hyperglycemia.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Control

Journal:

Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

doi: 10.1016/j.cellsig.2010.07.012

Figure Lengend Snippet: A. VEGF mRNA translation was studied by polysome assay. The shift of VEGF mRNA toward the heaviest fractions in diabetic kidneys indicates active translation, and is reversed by the AT2 but not the AT1 antagonist. Note that the distribution of GAPDH mRNA is unaffected by the various treatments. The figure is representative of experiments performed on kidneys from 3 individual mice. B. Association of hnRNP K with VEGF mRNA was studied by immunoprecipitation with anti-hnRNP K antibody followed by extraction of associated RNA associated and RT-PCR. Note that hnRNP K is constitutively associated with both VEGF and GAPDH mRNAs and that only the association with VEGF mRNA is increased in diabetic kidneys. The figure is representative of experiments performed on kidneys from 3 individual mice. **p<0.01, ns = not significant by ANOVA.

Article Snippet: Age- and sex-matched AT2 knockout mice (a generous gift from Dr Victor Dzau, Duke University) and their wild-type controls (129S6/SvEvTac, Jackson Laboratories, Bar Harbor, ME) received STZ as described above and were sacrificed 24 hours after onset of hyperglycemia.

Techniques: Immunoprecipitation, Extraction, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

doi: 10.1016/j.cellsig.2010.07.012

Figure Lengend Snippet: A. AT2 protein level was measured by immunoblot using a specific antibody. The lower panel shows combined data obtained on kidneys from 5 individual mice for each experimental condition. B. VEGF protein (B) and mRNA (C) levels were measured by immunoblot and RT-qPCR, respectively, on the same number of kidneys as in panel A. Identical experiments were performed for the protein and mRNA levels of fibronectin (D - E) and laminin β1 (F - G). **p<0.01, ns = not significant by ANOVA.

Article Snippet: Age- and sex-matched AT2 knockout mice (a generous gift from Dr Victor Dzau, Duke University) and their wild-type controls (129S6/SvEvTac, Jackson Laboratories, Bar Harbor, ME) received STZ as described above and were sacrificed 24 hours after onset of hyperglycemia.

Techniques: Western Blot, Quantitative RT-PCR

Journal:

Article Title: Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

doi: 10.1016/j.cellsig.2010.07.012

Figure Lengend Snippet: The diagram depicts a summary of the data obtained in this study. AT1 activation by hyperglycemia activates mTOR independently of Akt and leads to inactivation of eEF2K but is not sufficient to cause the dephosphorylation and activation of eEF2. Activation of AT2 stimulates the Akt-mTOR pathway that inactivates eEF2K and activates PP2A that dephosphorylates eEF2; AT2 also mediates hyperglycemia-induced increment in eEF1A. Activation of eEF2 and up-regulation of eEF1A stimulate elongation phase of mRNA translation, leading to increased translation of VEGF mRNA.

Article Snippet: Age- and sex-matched AT2 knockout mice (a generous gift from Dr Victor Dzau, Duke University) and their wild-type controls (129S6/SvEvTac, Jackson Laboratories, Bar Harbor, ME) received STZ as described above and were sacrificed 24 hours after onset of hyperglycemia.

Techniques: Activation Assay, De-Phosphorylation Assay