Differential expression of a V-type ATPase C subunit gene, Atp6v1c2, during culture of rat lung type II pneumocytes

Abstract

The lung alveolar epithelium consists of type I and type II pneumocytes. In vivo, the type II cell is the progenitor cell from which the type I cell originates. When freshly-isolated type II cells are cultured under conventional conditions they rapidly lose their phenotypic properties and attain characteristics of type I cells. Taking advantage of this transdifferentiation, we sought to identify genes that are differentially expressed during culture of rat type II cells. Using suppression subtractive hybridization (SSH), a vacuolar-type H+-ATPase (V-ATPase) C2 subunit gene (Atp6v1c2) was found to be enriched in freshly isolated rat type II cells compared to those cultured for 4 days. Northern blotting and reverse-transcription polymerase chain reaction (RT-PCR) confirmed the differential expression of Atp6v1c2 during in vitro culture of isolated type II cells. Expression ofAtp6v1c2 was significantly reduced early during in vitro culture: almost 90% reduction was observed after 24 h of incubation as determined by real-time PCR. In situ hybridization showed that Atp6v1c2 is expressed in both bronchiolar and alveolar lung epithelial cells, an expression pattern similar to that of surfactant protein B (SP-B). Multi-tissue Northern blotting revealed a unique tissue distribution with Atp6v1c2 expression limited to lung, kidney and testis. The presence and expression of Atp6v1c2 gene transcript isoforms, resulting from alternative splicing, were also investigated. Elucidation of differential expression of Atp6v1c2 in type II cells and further studies of its regulation may provide information useful in understanding the molecular mechanism underlying phenotypic and functional changes during transdifferentiation of alveolar epithelial cells.

Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase

“…Although these subunits (C1, C2-a, and C2-b) are all structurally different, their functional differences remain unclear. 12,23) Sun-Wada et al showed that V-ATPase containing C2-a or C2-b exhibited a lower K m value or coupling efficiency between ATP hydrolysis and proton transport, suggesting that the C isoforms are involved in the regulation of V-ATPase activity required in different tissues. 18) The human C2 isoform that we expressed using a cellfree protein synthesis system and characterized was the C2-b splice variant.…”

“…Isoform E1 is testis specific and forms a complex with the ubiquitous G1 subunit. Although C2 has 2 splice variants, we expressed and purified only C2-b for our studies, and we assume from previous studies in rats 23) that C2-b is functional during spermatogenesis (human and rat C2-b show 83% sequence identity). The a2 isoform is localized in the Golgi and it is known that the E1 and a2 isoforms are solely responsible for acrosome acidifica-tion in the testis.…”

“…C2-a is predominantly expressed in the lung, whereas C2-b is expressed in the kidney and testis. 18,23) Among the 3 G isoforms, G1 is found ubiquitously, whereas G2 is found in the brain and G3 in the kidney and inner ear. 20,21,24) Kidney, testis, inner-ear, osteoclast, brain, and lung require the isoform combinations of E2G3-a4-C2-H, E1G1-a2-C2-H, E2G3-a4-C1-H, E2G1-a3-C1-H, E2G2-a1-C1-H, and E2G1-a1-C2-H, respectively, for their specific functionality.…”

The vignette for V12N6 issue

“…A vacuolar-type H + -ATPase (V-ATPase) C2 subunit gene (Atp6v1c2) was found to be enriched in freshly isolated rat type II cells and almost 90% reduction was observed after 24 h of incubation. In situ hybridization showed that Atp6v1c2 is expressed in both bronchiolar and alveolar lung epithelial cells [8]. Elucidation of differential expression of Atp6v1c2 in type II cells provides information useful in understanding the molecular mechanism during transdifferentiation of alveolar epithelial cells.…”

Plasticity, Nuclear Diapause, and a Requiem for the Terminal Differentiation of Epithelia

“…9,12 Implied in this notion is mature epithelia, although not multipotent, have some degrees of freedom in reprogramming their chromatin and changing their phenotype in response to new somatic cues ( Figure 1A). Interconversions, for example, have been observed above the gastroesophageal junction, where stratified squamous epithelial cells become columnar, like the intestine, to form Barrett's esophagus 13 ; in type B intercalated cells from the renal collecting duct that change into type A intercalated cells; 14 -16 in type 2 pneumocytes that transdifferentiate into type 1 pneumocytes in the lung; 17,18 in neural retinal cells that morph into pigmented epithelia; 19 in retinal pigmented cells that become lens epithelia; 20,21 in lactotrophs that interconvert to somatotrophs in the pituitary gland; 22 in pancreatic exocrine cells that become hepatocytes; [23][24][25] and in hepatocytes that transdifferentiate into insulin producing cells. 26 -29 All of the drivers underlying these conversions are not understood, but a couple of examples are illustrative.…”