Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues

Abstract

Cyclin D1 is a key cell-cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue-dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non-dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.

Fig. 1

Expression of cyclin D1 in several mouse tissues. (a) Representative Western blot analysis of cyclin D1 in mouse tissues. (b) The loading and transfer of equal amounts of proteins were confirmed after transfer to PVDF membrane by staining the membranes with Red Ponceau.

Fig. 2

Distribution of cyclin D1 in several mouse tissues. (a) Epithelial cells of terminal bronchioles: strong nuclear immunostaining of cyclin D1 contrasts with faint cytoplasmic staining; 450×. (b) Salivary duct cells: intense cytoplasmic staining of cyclin D1; 450×. (c) Intestine, cyclin D1 localized in the cytoplasm of enterocytes; 650×. (d) Kidney: cytoplasmic staining is present in the cytoplasm of tubular epithelium cells; 650×. (e) Cerebral cortex of adult mouse brain: cytoplasmic staining of cyclin D1 in several neurons; 150×. (f) Nervous ganglia of embryo brain: strong nuclear immunostaining can be seen in neuronal nuclei; 200×. (g) Grey matter of medulla oblongata of mouse embryos: strong cyclin D1 immunostaining was present in almost all nuclei; 200×. (h) No immunoreactivity present in a representative negative control of adult brain; 450×.

Fig. 3

Cyclin D1 localization in cell compartment: cyclin D1 nuclear/cytoplasmic ratio in cells of several mouse tissues. All values were expressed as mean ± standard error of mean (SEM).

Fig. 4

Cyclin D1 subcellular localization by immunoelectron microscopy assay. (a) Prevalent localization of cyclin D1 in the cytoplasm of intestinal enterocytes with a consequent low nuclear/cytoplasmic ratio. Abbreviations: N, nucleus; C, cytoplasm; M, mitochondrion; 20 000×. (b) Localization of cyclin D1 in the space of nuclear pore in a kidney cell indicating the shift between nuclear and cytoplasmic compartments; 20 000×. (c) A higher magnification showing the positivity of cyclin D1 in the nuclear pore; 90 000×.