Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37°C and 4°C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.

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The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D 3 in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D 3 in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.A primary function of vitamin D is to markedly increase intestinal absorption of calcium and phosphate (1). During the 1950s, this absorption was shown to be primarily an active calcium transport process and an independent active phosphate transport process (2, 3). With the discovery of the vitamin D endocrine system came the understanding that this process was directly stimulated by the hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) (4). Because this hormonal form of vitamin D is regulated in response to the need for calcium, it became clear that the endogenous factor discovered by Nicolaysen and Egg-Larsen is the vitamin D endocrine system producing the final vitamin D hormone, 1,25-(OH) 2 D 3 (4, 5). There is still debate whether vitamin D further influences the diffusional component of calcium absorption, taking place at high intestinal levels of calcium (6, 7).The molecular mechanism underlying active calcium transport in response to vitamin D began unraveling with the discovery of calbindin D 9k by Wasserman and colleagues (8). Schachter and colleagues (9) also found a transporter responsive to vitamin D. Others have reported that vitamin D stimulates the basal lateral membrane calcium ATPase believed to be a calcium transporter (10, 11). These components have been put together in a diagrammatic fashion to present the current hypothesis of how 1,25-(OH) 2 D 3 stimulates active intestinal calcium absorption. TRPV6 is a calcium channel protein clearly induced by 1,25-(OH) 2 D 3 (12). Calcium entering through this channel is believed to associate with calbindin D 9k , which serves as a shuttle for calcium, presenting it to the basal lateral membrane calcium ATPase. This step provides the energy input for the transfer of calcium against a concentration gradient. All three of these genes are clearly under the influence of the active form of vitamin D (13). Unfortunately not all evidence is currently in support of this hypothesis. Transgenic mice in which calbindin D 9k has been eliminated have shown that calbindin D 9k is not required for 1,25-(OH) 2 D 3 -stimulated intestinal calcium absorption (14, 15). Fo...