Deletion of ATG5 shows a role of autophagy in salivary homeostatic control - PubMed (original) (raw)

doi: 10.1177/0022034513499350. Epub 2013 Jul 24.

H H Lin, A M Chibly, W Zhang, X Sun, C-H Chen, P Flodby, Z Borok, R Wu, D Arnett, R R Klein, D K Ann, K H Limesand

Affiliations

Deletion of ATG5 shows a role of autophagy in salivary homeostatic control

M Morgan-Bathke et al. J Dent Res. 2013 Oct.

Abstract

Autophagy is a catabolic pathway utilized to maintain a balance among the synthesis, degradation, and recycling of cellular components, thereby playing a role in cell growth, development, and homeostasis. Previous studies revealed that a conditional knockout of essential member(s) of autophagy in a variety of tissues causes changes in structure and function of these tissues. Acinar cell-specific expression of knocked-in Cre recombinase through control of aquaporin 5 (Aqp5) promoter/enhancer (Aqp5-Cre) allows us to specifically inactivate Atg5, a protein necessary for autophagy, in salivary acinar cells of Atg5(f/f);Aqp5-Cre mice. There was no difference in apoptotic or proliferation levels in salivary glands of Atg5/Cre mice from each genotype. However, H&E staining and electron microscopy studies revealed modestly enlarged acinar cells and accumulated secretory granules in salivary glands of Atg5(f/f);Aqp5-Cre mice. Salivary flow rates and amylase contents of Atg5/Cre mice indicated that acinar-specific inactivation of ATG5 did not alter carbachol-evoked saliva and amylase secretion. Conversely, autophagy intersected with salivary morphological and secretory manifestations induced by isoproterenol administration. These results identified a role for autophagy as a homeostasis control in salivary glands. Collectively, Atg5(f/f);Aqp5-Cre mice would be a useful tool to enhance our understanding of autophagy in adaptive responses following targeted head and neck radiation or Sjögren syndrome.

Keywords: animal models; apoptosis; isoproterenol; proliferation; salivary gland.

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Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.

Figure 1.

Conditional inactivation of ATG5 fails to affect salivary apoptosis and proliferation. (A-F) Salivary gland tissues were collected from four- to six-week-old naïve Atg5+/+, Atg5+/+;Aqp5-Cre, Atg5f/+, Atg5f/+;Aqp5-Cre, Atg5f/f, and Atg5f/f;Aqp5-Cre mice, formalin-fixed and paraffin-embedded. Percentages of positive cells were determined from the means of 3 to 5 images per mouse. (A) Quantification of positive cleaved caspase-3 staining of naïve PAGs. (B) Positive cleaved caspase-3 staining of naïve SMGs. (C) Positive PCNA staining of naïve PAG acinar cells. (D) Positive PCNA staining of naïve PAG ductal cells. (E) Positive PCNA staining of naïve SMG acinar cells. (F) Positive PCNA staining of untreated SMG ductal cells. (A-F) p > .05; n ≥ 4 per genotype. (G) Representative images of cleaved caspase-3 staining. (H) Representative images of positive PCNA staining. Scale bar: 50 µm.

Figure 2.

Figure 2.

Conditional knockout of Atg5 causes enlarged salivary acinar cells. (A) Salivary gland tissues were collected from four- to six-week-old naïve Atg5+/+, Atg5+/+;Aqp5-Cre, Atg5f/+, Atg5f/+;Aqp5-Cre, Atg5f/f and Atg5f/f;Aqp5-Cre mice, formalin-fixed and paraffin-embedded for H&E studies. (B) Dissected PAGs and SLGs from Atg5f/f and Atg5f/f;Aqp5-Cre mice were fixed in glutaraldehyde and analyzed by transmission electron microscopy (TEM). Two representative TEM images with different magnifications (1100x and 2700x) from each gland are shown. (C) Frozen sections of dissected SLGs from Atg5f/+;Aqp5-Cre and Atg5f/-; Aqp5-Cre mice were stained with an anti-MUC5B antibody and visualized with an Alexa Fluor 488-conjugated secondary antibody. F-actin and nuclei were respectively stained with TRITC-conjugated phalloidin and DAPI. Scale bar: 20 µm. (D) Salivary glands of naïve male mice aged to 6 mos (upper two panels) and 18 mos (lower two panels) were analyzed for changes in structure using H&E slides. Magnification: 40x. Arrows indicate hypertrophic cells. Scale bar: 50 µm.

Figure 3.

Figure 3.

Inactivation of Atg5 in salivary acinar cells does not affect carbachol-stimulated salivary gland function. Stimulated salivary flow rates were normalized to Atg5+/+ mice for their respective gender. (A) Carbachol-stimulated saliva was collected from four- to six-week-old female (upper panel) and male (lower panel) Atg5+/+, Atg5+/+;Aqp5-Cre, Atg5f/+, Atg5f/+:Aqp5-Cre, Atg5f/f and Atg5f/f;Aqp5-Cre mice. p > .01; n ≥ 10 per genotype. (B) Representative image of Experion Chip analyses of saliva collected from four- to six-week-old carbachol-treated female and male Atg5/Cre mice with 6 different genotypes and analyzed for percentage amylase determined by the 54- to 56-kD band. n ≥ 10 per genotype. (C) The amylase levels from stimulated saliva from female (left panel) and male (right panel) Atg5+/+, Atg5+/+;Aqp5-Cre, Atg5f/+, Atg5f/+:Aqp5-Cre, Atg5f/f and Atg5f/f;Aqp5-Cre mice (one representative profile is shown in B) were summarized. p > .01; n ≥ 10 per genotype.

Figure 4.

Figure 4.

Autophagy intersects with Ipr-triggered morphological and functional manifestations. (A) Four- to six-month-old male mice were given daily injections (i.p.) of Ipr or vehicle for 10 consecutive days. Salivary tissues were collected and sections stained for hematoxylin and eosin (H&E) on the day following the last injection: representative H&E of PAGs, SMGs, and SLGs from untreated Atg5f/f mice (top panels); representative H&E of PAGs, SMGs, and SLG from Ipr-injected Atg5f/f mice (2nd-row panels); representative H&E of PAGs, SMGs, and SLGs from untreated Atg5f/f;Aqp5-Cre mice (3rd-row panels); and representative H&E of PAGs, SMGs, and SLGs from Ipr-injected Atg5f/f;Aqp5-Cre mice (bottom panels). Scale bar: 50 µm. (B) The effect of chronic Ipr administration on salivary glands of autophagy-deficient mice. Atg5f/−;Aqp5-Cre (KO) and Atg5f/+;Aqp5-Cre (WT) mice were subjected to Ipr injections for 10 days as in (A). Western blot analyses of PAG and SMG tissue lysates were performed with the indicated primary antibodies. NT; no treatment control. The underlying cause for the abnormal migration of ATG5-ATG12 and p62/SQSTM1 in PAGs of Ipr-injected mice, albeit reproducible, was unclear. (C) PAGs and SLGs were harvested from vehicle- and Ipr-injected Atg5f/f;Aqp5-Cre (Atg5-/-) and Atg5f/f mice at 30 min post-administration. Dissected tissues were examined by TEM. Quantification of secretory granules in PAGs and SMGs per field of TEM images (magnification 1,100x; n = 25; one representative image is shown in Appendix Fig. 1) of vehicle- and Ipr-injected Atg5f/f;Aqp5-Cre (Atg5-/-) and Atg5f/f mice is shown as mean ± SD.

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