Mechanical strain regulation of the Chicken glypican-4 gene ...

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entered the ESG (Fig. 3B). Two hours after the egg entered the ESG (Fig. 3C) some of the epithelium cells started to express the GPC-4 gene. Gradually, more epithelium cells expressed the GPC-4 gene, and 8 h after the entry of the egg into the ESG (Fig. 3D) – at the time when the shell is being rapidly formed – most of the cells expressed the GPC-4 gene. A high level of expression was observed during all the time of massive calcification (Fig. 3E – 12 h after the entry of the egg into the ESG). One hour before oviposition (Fig. 3F), at the phase of eggshell completion, a massive reduction in the gene expression was observed. The epithelium of the ESG consists of two different cell types: the pseudostratified epithelium (PE) facing the lumen and inner glandular epithelium (GE). At higher magnification, the GPC-4 gene was found to be expressed exclusively by the GE cells and not by the PE cells, regardless of the time the egg had resided in the ESG (Fig. 4). Effect of mechanical stress on the GPC-4 gene expression Insertion of an egg-shaped endotracheal tube into the ESG 3 h after oviposition, at a time when the GPC-4 gene is not naturally expressed (Fig. 5A) caused gene induction. The expression of the GPC-4 gene was strain-dependent: insertion of a small-size tube (with a volume of 20 cm 3 ) caused a lower induction of GPC-4 gene expression (Fig. 5B) than observed with the larger tube (60 cm 3 ; Fig. 5C). Moreover, removing the mechanical strain by expulsion of the egg close to the time of its entry into the ESG and thus causing premature oviposition, attenuated GLC-4 gene expression (Fig. 6A), compared with the level of expression in the control laying hen (Fig. 6B). No change in �-actin gene expression was observed in ESG sections 3 and 12 h after oviposition or after mechanical strain application at the time of major increase in the CPC-4 expression, ruling out the possibility of variation in tissue processing and RNA preservation (Fig. 7). Expression of the GPC-4 gene depends on mechanical strain, not only in the ESG but also in other parts of the oviduct. In the magnum, the gene is expressed only when the egg resides within this part of the oviduct and mechanical strain is imposed (Fig. 8). No effect of mechanical strain was observed in the sexually immature pre-laying pullet (data not shown). Expression GPC-4 gene in various tissues 10
The expression of the GPC-4 gene in a variety of tissues was examined by RT-PCR (Fig. 9). In addition to the high levels of the GPC-4 gene that were expressed in the ESG , high levels were also observed in the liver, pancreas and kidneys. The expression of the GPC- 4 gene in these tissues was unaffected by the daily egg cycle (data not shown). DISCUSSION The avian GPC-4 gene exhibits high homology with the mammalian ones and presents highly conserved sequences such as the glypican signature and the GPI-binding site in the same locations as the mammalian homologues. Moreover, the conserved locations of the cystein residues suggest similar three-dimensional structures (Fig. 2). To date, GPC-4 is the second member of the glypican family identified in the chicken [the first being GPC-1 (29)], which suggests that, similarly to mammals, avian species may contain more members of this family. In the present study we demonstrated for the first time that the avian GPC-4 gene is expressed in the ESG in a circadian fashion and is probably regulated by mechanical strain. This hypothesis is supported by the following observations: A- The gene is expressed in the ESG only when an egg resides in the ESG and imposes a mechanical strain (Fig. 3); B- Removal of the mechanical strain caused reduction in the gene expression (Fig. 6); C- Artificial application of the mechanical strain caused induction of the GPC-4 expression that was related to the level of the strain (Fig. 5); and D- the GPC-4 gene was induced by mechanical strain in other parts of the oviduct too (Fig. 8). In the ESG the GPC-4 was expressed only by the GE cells and not by the cells facing the lumen (Fig. 4). Previously (24) we demonstrated that OPN expressed by the PE cells was regulated by the mechanical strain while calbindin, a calcium-binding protein expressed by the GE cells in a circadian fashion, was not. Moreover, the α1 subunit of the Na-K ATPase that was expressed by both cell types was regulated by mechanical strain only in the PE cells, whereas in the GE cells it was regulated by the calcium flux (25). Thus GPC-4 is the first example of a mechanicalstrain-dependent gene expressed by the GE cells. This suggests that the mechanical strain signal that is first sensed by the cells facing the lumen had to be transduced to the inner cell layer either directly or mediated by factor(s) from the pseudostratified epithelium. The expression of other proteoglycan genes such as versican, biglycan, perlecan and 11
Page 1 and 2: AJP-Regu Articles in PresS. Publish
Page 3 and 4: Abbreviations ESG - avian eggshell
Page 5 and 6: about 10% of the total body calcium
Page 7 and 8: for 1 min, 60°C for 1 min and 68°
Page 9: RESULTS Identification of the chick
Page 13 and 14: to become part of the assembly of p
Page 15 and 16: 9. Clark EA, King WG, Brugge JS, Sy
Page 17 and 18: 30. Numaguchi K, Eguchi S, Yamakawa
Page 19 and 20: LEGENDS TO FIGURES Fig. 1. The asse
Page 21 and 22: 1. 2. 3. 4. 81 bp PCR 482 bp PCR 96
Page 23 and 24: Figure 3
Page 29: Figure 9

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