Calcium, calcium-sensing receptor and growth control in the colonic mucosa

Abstract

A role for calcium in epithelial growth control is well-established in the colon and other tissues. In the colon, Ca²+ "drives" the differentiation process. This results in sequestration of β-catenin in the cell surface / cytoskeletal complex, leaving β-catenin unavailable to serve as a growth-promoting transcription enhancer in the nucleus. The signaling events that lead from Ca²+ stimulation to differentiation are not fully understood. A critical role for the extracellular calcium-sensing receptor (CaSR) is assumed, based on CaSR localization to the differentiating epithelial cells in the normal colonic mucosa (upper half of the crypt and crypt surface), decreased CaSR expression in colon carcinoma, and the results from in vitro studies with colonic epithelial cell lines. While Ca²+ is well-accepted as a growth-regulating agent in the colon, suppression of cell proliferation is not complete. At least part of the reason for this is the inherent variability in Ca²+ responsiveness among individual epithelial cells. Of interest, colon epithelial cells that are resistant to the growth-regulating activity of Ca²+ alone are still responsive to Ca²+ in conjunction with other transition metals. Whether a multi-mineral approach will, ultimately, prove to be more effective than Ca²+ alone as a colon cancer chemopreventive agent remains to be seen, but certainly worth investigating.

Figures

Fig. 1

CaSR expression in normal colonic epithelium and in colon carcinoma. A and B. In the normal crypt, there is intense staining of the differentiated cells in the upper part of the crypt. C. In an area of moderately-differentiated tumor, weak and variable staining is observed. D. There is essentially no staining of isolated cells at the invasive front. Likewise, there is little or no staining of invasive cells even where there is rudimentary glandular structure. E and F. In areas of histologically undifferentiated tumor (no glandular structure evident; only sheets of undifferentiated tumor cells), there is no CaSR staining (See Chakrabarty et al. 2003; for details).

Fig. 2

Relationship between CaSR staining and ß-catenin staining. A: CaSR and B: ß-catenin: Where CaSR is expressed in the tissue, ß-catenin staining is strongly associated with the cell surface. Where CaSR is not expressed, intense cytoplasmic/nuclear ß-catenin staining is seen. Arrows in each panel demonstrate area of marked transition. (See Bhagavathula 2007 for details).

Fig. 3

Relationship between CaSR staining and Ki67 reactivity. A: CaSR and B: Ki67: Where CaSR is expressed (normal glandular structure), Ki67 staining is sporadic. Where CaSR is not expressed (tumor), a high percentage of cells are Ki67-positive.

Fig. 4

E-cadherin and ß-catenin expression in Ca2+-responsive and non-responsive CBS cells: Upper panels: Confocal fluorescence microscopy showing E-cadherin and ß-catenin staining patterns in Ca2+-responsive and non-responsive CBS cells. In the absence of extracellular Ca2+, diffuse cytoplasmic staining is observed with both proteins in both populations. In response to Ca2+, peripheral staining is observed in the Ca2+-responsive cells but the staining pattern in the non-responsive cells for both proteins is similar to what is observed in the absence of Ca2+. Lower panels: Cell fractionation studies showing relative distribution of the two proteins between cytosolic/nuclear fraction and membrane fraction in the absence and presence of Ca2+ (See Bhagavathula 2007 for details).

Fig. 5

Effect of extracellular Ca2+ on TCF-4 transcription in Ca2+-responsive and non-responsive CBS cells. Upper panel: TCF-4 transcription was measured using the dual luciferase reporter assay, and results were expressed as percent inhibition of CTAA in the presence of extracellular Ca2+ relative to control cells cultured in the absence of Ca2+. Lower panel: Cells were grown under Ca2+-free or Ca2+-containing conditions for 2 days. Lysates from these cells were probed for c-myc and cyclin D1 expression by Western blot analysis. (See Bhagavathula 2007 for details).

Fig. 6

Effects of a multi-mineral-rich, red marine algae extract on proliferation and differentiation in Ca2+-responsive and non-responsive CBS cells. Upper-left panel: Cells were treated with the mineral-rich extract or with Ca2+ alone (calcium chloride) and cell numbers were determined after 2 days of incubation. Upper-right panel: Morphology: Cells were stained with hematoxylin and eosin. Lower-left panel: Confocal immunofluorescence microscopy - E-cadherin. Lower-right panel: Western blot for E-cadherin. Parent cells demonstrated reduced growth and differentiation in response to either treatment. The Ca2+-non-responsive cells demonstrated growth reduction and differentiation in response to the multi-mineral product, but did not respond to Ca2+ alone (see Aslam 2009 for details).