Functional expression of PEPT2 and its regulation in alveolar epithelial cells

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1 Workshop on Drug Transporters in the Lungs TCD, Dublin, IRL, Sept , 216 Functional expression of PEPT2 and its regulation in alveolar epithelial cells Mikihisa Takano, Ph.D. Graduate School of Biomedical & Health Sciences, Hiroshima University, Japan 1 Functional expression of PEPT2 and its regulation in alveolar epithelial cells 1) Rat primary cultured alveolar epithelial cells (Type II cells, Type I-like cells) 2) Human distal lung epithelial cell line NCI-H441 1

Functional expression of PEPT2 and its regulation in alveolar epithelial cells

Type I-like cells) Expression and function of PEPT2 during

Characteristics of primary cultured alveolar epithelial cells (type II and

dish/2 days) Phase-contrast micrographs Confocal laser scanning micrographs

2 Functional expression of PEPT2 and its regulation in alveolar epithelial cells 1. Study with rat primary cultured alveolar epithelial cells (Type II cells, Type I-like cells) Expression and function of PEPT2 during transdifferentiation of primary cultured alveolar epithelial cells Characteristics of primary cultured alveolar epithelial cells (type II and type I-like cells) we have been using Type II cells (5 x 1 6 cells/35 mm dish/2 days) Phase-contrast micrographs Confocal laser scanning micrographs (CLSM) of lamellar bodies in type II cells Type I-like cells (2 x 1 6 cells/35 mm dish/6 days) LysoTracker Red Nile Red 2

Immunostaining of ABCA3 and caveolin-1 protein in primary cultured rat

PCR and Microarray analyses Type II Type I-like The expression of about 12

respectively, along with transdifferentiation (total probes 35).

drastically during transdifferentiation.

caveolin-1 increased markedly, while type II marker mrnas such as CINC-1

3 Immunostaining of ABCA3 and caveolin-1 protein in primary cultured rat alveolar epithelial cells Type II cell Type I-like cell ABCA3 (type II cell marker) Caveolin-1 (type I cell marker) Change in mrna expression profile during transdifferentiation of type II cells into type I-like cells Real-time PCR and Microarray analyses Type II Type I-like The expression of about 12 mrnas increased more than 2-fold and decreased to less than 1/2, respectively, along with transdifferentiation (total probes 35). In both methods, we observed that the expression level of many mrnas changed drastically during transdifferentiation. For example, in Real-time PCR analysis, type I marker mrnas such as caveolin-1 increased markedly, while type II marker mrnas such as CINC-1 decreased markedly after transdifferentiation. Primary cultured alveolar epithelial cells we have been using have type II and type I cell phenotype, respectively. 3

4 Background When we started this study, it was reported that PEPT2 was expressed in type II cells using in situ hybridization and immunohistochemistry, and that PEPT2 in type II cells could transport a fluorophore conjugated dipeptide derivative under ex vivo conditions (Groneberg et al., 21, 22 ). However, the relationship between the transdifferentiation of alveolar epithelial cells and functional expression of PEPT2 was not clear. In addition, there was little information concerning the transport characteristics of PEPT2 at the alveolar cell level. Therefore, using these primary cultured alveolar type II and type I-like epithelial cells, we studied and compared the expression and function of PEPT2. Expression of PEPT2 mrna during transdifferentiation of alveolar epithelial cells Transport function of PEPT2 during transdifferentiation of alveolar epithelial cells (Gly-Sar uptake) Expression of Pept2 mrna (% of Day ) Day Day 1 Day 2 Day 6 [ 3 H]Gly-Sar uptake (nmol/mg protein/hr) Control Cefadroxil Day 1 Day 2 Day 4 Day 6 The expression and function of PEPT2 would decrease during transdifferentiation of alveolar epithelial cells. 4

5 Expression of PEPT2 protein in type II and type I-like alveolar epithelial cells Time-course Effect of extracellular ph on glycylsarcosine (Gly-Sar) uptake in alveolar epithelial cells 1.6 [ 3 H] Gly-Sar uptake (nmol/mg protein) ph = Time (min) ph = 7.4 type II type I-like ph = 6. ph = 7.4 5

6 ph-dependence of Gly-Sar uptake in alveolar type II cells Concentration-dependent effect of cefadroxil on Gly-Sar uptake in alveolar epithelial cells (ph 6.) 3. type II.3 type I-like [ 3 H] Gly-Sar uptake (nmol/mg protein) IC5 value = 4.3 μm Cefadroxil ( M) Cefadroxil ( M) P <.1 6

transdifferentiation, and PEPT2 is almost completely lost in type I cells.

7 Concentration-dependence of Gly-Sar uptake in alveolar type II cells (ph 6.) Km = 72. μm Summary 1 PEPT2 is functionally expressed in primary cultured alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 is almost completely lost in type I cells. So, primary cultured type II cells are a good in-vitro model to study alveolar PEPT2 function. However, they are not suitable for long term study, because of phenotype change during culture. Therefore, alveolar epithelial cell line naturally and stably expressing PEPT2 would be useful to study the function, expression, and its regulation. 7

8 Gly-Sar uptake in widely used alveolar epithelial cell lines, RLE-6TN and A549 Effect of extracellular ph on Gly-Sar uptake in RLE 6TN and A549 cells Effect of cefadroxil on Gly-Sar uptake in RLE 6TN and A549 cells.8 RLE-6TN cells.15 A549 cells [ 3 H] Gly-Sar uptake (nmol/mg protein) Cefadroxil ( M) Cefadroxil ( M) PEPT2 is not functionally expressed in RLE-6TN and A549 cells, and these cells are not good in-vitro models to study alveolar PEPT2. 8

9 Functional expression of PEPT2 and its regulation in alveolar epithelial cells 2. The expression and function of PEPT2 in human distal lung epithelial cell line NCI-H441 Morphology of H441 cell monolayers and lamellar body formation in H441 (day 13) and A549 cells Epithelial-like morphology H441 H441 Dome-like structure H441 A549 9

The mrna expression of type II cell markers (SP-C and

day 7) 3 2 1 ABCA3 Expression (% of day 7) 4 3 2 1 day 7

Expression (% of day 7) 4 3 2 1 PEPT1 (expected size) PEPT2

10 The mrna expression of type II cell markers (SP-C and ABCA3), PEPT2 and PEPT1 in H441 cells SP-C Expression (% of day 7) ABCA3 Expression (% of day 7) day 7 day 9 day 11 day 13 day 7 day 9 day 11 day 13 PEPT2 Expression (% of day 7) PEPT1 (expected size) PEPT2 day 7 day 9 day 11 day 13 PEPT2 PEPT1 H441 cells would have alveolar type II cell-like phenotype. The expression of PEPT2 protein in H441 and A549 cells Immunostaining H441 A549 Western Blotting 1

11 Effect of cefadroxil on Gly-Sar uptake in H441 cells [ 3 H]-Gly-Sar uptake (pmol/mg protein) [ 3 H]-Gly-Sar uptake (pmol/mg protein) o C Time (min) 4 o C Time (min) [ 3 H]-Gly-Sar uptake (% of control) Concentration dependence * * * * Conc. of cefadroxil (mm) : Control : Cefadroxil ph-dependence of cefadroxil-sensitive (PEPT2- mediated) Gly-Sar uptake in H441 and A549 cells H441 A549 11

12 Concentration-dependence of Gly-Sar uptake in H441 cells (ph 6.5) Km = μm TEER and PEPT2-mediated transcellular transport of cephalexin across H441 cell monolayers TEER Transepithelial transport (Cefadroxil-sensitive) A-to-B B-to-A TEER values of A549 and RLE-6TN cell monolayers were much lower than H441 cells, 31 and 78 ohm cm 2, respectively. 12

13 Summary 2 PEPT2 is functionally expressed in H441 cells, making the cell line a good in-vitro model to study PEPT2 function and its regulation in human distal lung. Especially, H441 cells form electrically tight monolayers, and therefore can be used for transcellular transport studies. PEPT2 and innate immune response In primary human upper airway epithelial culture cells, it is reported that γ-ie-dap (γ-d-glutamyl-mesodiaminopimelic acid), a bacterial dipeptide, is taken up by PEPT2, and induces the innate immune response in the NOD1 (nucleotide-binding oligomerization domain 1) - -dependent manner (Peter W. Swaan et al., Am J Respir Cell Mol Biol Vol 39. pp , 28). 13

14 Possible induction of innate immune response by γ-ie-dap in alveolar type II cells Preliminary data Expression of NOD1 mrna in H441 cells 14

15 Preliminary data Effect of γ-ie-dap on mrna expression of IL-6 and IL-8 in H441 cells IL-6 IL-8 % of control γ-ie-dap(-) γ-ie-dap(+) % of control γ-ie-dap(-) γ-ie-dap(+) 4 hr 24 hr 4 hr 24 hr Summary 3 (Preliminary) PEPT2- and NOD1-dependent immune response may occur in alveolar type II epithelial cells, which may serve as one of the hostdefense systems in the distal lungs. 15

Supplementary Figure 1. Markedly decreased numbers of marginal zone B cells in DOCK8 mutant mice Supplementary Figure 2.

Supplementary Figure 1. Markedly decreased numbers of marginal zone B cells in DOCK8 mutant mice. Percentage of marginal zone B cells in the spleen of wild-type mice (+/+), mice homozygous for cpm or pri