Ramesh K. Wali

1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.

The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroids effects on colonocyte calcium transport and proliferation.

Simultaneous measurement of angular and spectral properties of light scattering for characterization of tissue microarchitecture and its alteration in early precancer

We present a novel instrument to measure the spectral, angular, azimuthal, and polarization dependence of light backscattered by living biological tissues, thus providing the most comprehensive description of the light scattering to obtain unique quantitative information about the microarchitecture of living cells and tissues. We show the potential of this technique to characterize and diagnose early premalignant changes in the epithelia. In studies with a rodent model of colon carcinogenesis, we show that several parameters obtained using this technique, such as the number density of red blood cells in the capillary network immediately underlying the epithelium, the fractal dimension of the tissue, and the average roundness of subcellular structures, are significant for detection of precancerous changes at a very early stage of the carcinogenic process, at which no other histological or molecular markers have been identified.

The transcriptional repressor SNAIL is overexpressed in human colon cancer.

Overexpression of the transcriptional repressor, SNAIL, has been implicated in the pathogenesis of a number of malignancies; however, there are no previous reports on the role of SNAIL in colorectal cancers (CRCs). We, therefore, evaluated human CRC specimens for the presence of the SNAIL protein. Immunohistochemical studies were performed using samples obtained from archived CRC paraffin blocks and a tissue array. Tissue sections were probed with a polyclonal antibody to human SNAIL and scored by a gastrointestinal pathologist. SNAIL was not detectable in uninvolved mucosa, but immunoreactivity was evident in 78% of tumors. SNAIL protein expression did not correlate with subsite location or gender, however, SNAIL-positive tumors had an older mean age (58.9 ± 12.7 versus 49.8 ± 127; P = 0.028). Furthermore, there was a trend that CRCs with metastatic ability more frequently overexpressed SNAIL (100 versus 65%; P = 0.11). In conclusion, we demonstrate, for the first time, that SNAIL is upregulated in human colon cancer, which potentially may have significance in control of metastasis and possibly serve as a target for chemopreventive agents.

Coherent backscattering spectroscopy.

Coherent backscattering (CBS) of light in random media has been previously investigated by use of coherent light sources. Here we report a novel method of CBS measurement that combines low spatial coherence, broadband illumination, and spectrally resolved detection. We show that low spatial coherence illumination leads to an anomalously broad CBS peak and a dramatic speckle reduction; the latter is further facilitated by low temporal coherence detection. Thus CBS can be observed in biological tissue and other media that previously were beyond the reach of conventional CBS measurements. We also demonstrate, for the first time to our knowledge, spectroscopic analysis of CBS. CBS spectroscopy may find important applications in probing random media such as biological tissue in which depth-selective measurements are crucial.

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non‐genomic actions of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(−/−) osteoblasts isolated from wild‐type and VDR null mice to study the increase in intracellular calcium ([Ca2+]i) and activation of protein kinase C (PKC) induced by 1,25(OH)2D3. Within 1 min of 1,25(OH)2D3 (100 nM) treatment, an increase of 58 and 53 nM in [Ca2+]i (n = 3) was detected in VDR(+/+) and VDR(−/−) cells, respectively. By 5 min, 1,25(OH)2D3 caused a 2.1‐ and 1.9‐fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated‐MBP4–14 in VDR(+/+) and VDR(−/−) osteoblasts. The 1,25(OH)2D3‐induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)2D3 treatment resulted in the same degree of translocation of PKC‐α and PKC‐δ, but not of PKC‐ζ, from cytosol to plasma membrane in both VDR(+/+) and VDR(−/−) cells. These experiments demonstrate that the 1,25(OH)2D3‐induced rapid increases in [Ca2+]i and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)2D3 in osteoblasts.

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

Low-coherent backscattering spectroscopy for tissue characterization

Although the phenomenon of coherent backscattering (CBS) in nonbiological media has generated substantial research interest, observing CBS in biological tissue has been extremely difficult. Here we show that the combination of low-spatial-coherence, broadband illumination, and low-temporal-coherence, spectrally resolved detection significantly facilitates CBS observation in biological tissue and other random media with long-transport mean-free path lengths, which have been previously beyond the reach of conventional CBS investigations. Furthermore, we demonstrate that depth-selective, speckle-free, low-coherent backscattering spectroscopy has the potential to diagnose the earliest, previously undetectable, precancerous alterations in the colon by means of probing short light paths.

Increased microvascular blood content is an early event in colon carcinogenesis

Background: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. Methods: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. Results: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. Conclusion: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.

1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.

Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca(2+)-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl phorbol 13-acetate and 1,25(OH)2D3 activated endogenous PKC, as assessed by inhibition of myristoylated alanine-rich C kinase substrate back-phosphorylation by exogenous PKC. These studies indicate that PKC-alpha, -delta, and/or -epsilon likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.