We assessed whether consuming UV light-exposed mushrooms (UVMs) would offset the seasonal decline in circulating total 25-hydroxyvitamin D (25OHD), an index of vitamin D status. During late fall and winter, 41 adults (19 M/22 F, age 43 ± 11 y, BMI 29.8 ± 5.9 kg/m2, mean ± SD) were randomized to consume either 84 g of fresh Agaricus bisporus twice/d (produced to contain 400 IU of vitamin D2/serving; 800 IU/d total) or 1 tsp of breadcrumbs twice/d (Control) while continuing to consume their self-chosen diets. At baseline and week 6, fasting serum 25OHD2, 25OHD3, and total 25OHD were measured. Mushrooms were sampled weekly and vitamin D2 content measured. From the intent-to-treat analysis (Mushroom group, n = 20, and Control group, n = 21), 25OHD2 increased and 25OHD3 decreased in the Mushroom group over winter months compared to Control with no differences in the decrease in total 25OHD between groups. Unexpectedly, only 67% of the UVMs contained vitamin D2. Post hoc subgroup assessment indicated that participants consuming UVMs (n = 11) had increased 25OHD2 and a greater decline in 25OHD3 compared to subgroups consuming mushrooms without vitamin D2 (n = 9) and Control (n = 21), with no differences in the decrease in total 25OHD among subgroups. Consuming UVMs increased serum 25OHD2 but did not prevent a seasonal decline in vitamin D status due to a greater decrease in 25OHD3.

Abstract Intestinal stem cells (ISC) are viewed as the origin of colorectal cancer (CRC), but the early stages of carcinogenesis are unclear. We hypothesize that early, non-transforming mutations reprogram ISCs into a preneoplastic state that increases the risk of transformation. We performed single-cell RNA sequencing (scRNA-seq) on colon epithelial cells from Car1-Cre (CAC) mice, which express Cre specifically in colon epithelial cells. CAC mice were crossed to those with floxed oncogene alleles to generate mice with one (Apc+/-, AC) or two (Apc+/-; KrasG12D/wt, AKC) non-transforming mutations. AC mice had normal crypt morphology and low adenoma incidence, while AKC mice exhibited crypt elongation by 4 weeks and multiple adenomas by 8-10 weeks. Cells from distal colonic crypts (n=7) and from tumors (n=3) representing phenotypes ranging from normal to malignant were profiled using the 10X Genomics Chromium. Processing and clustering with Cell Ranger and Seurat yielded 58,906 cells from crypts and 36,327 from tumors. Initial clustering identified epithelial, immune, endothelial, and cancer groups. The epithelial group was subclustered and visualized by UMAP. Cells from CAC crypts followed the classic differentiation path: Stem, Progenitor, Transit-amplifying, Absorptive epithelial cell, Goblet, Tuft, and Enteroendocrine. Stem cell clusters were classified as Normal (CAC), Abnormal 1 (young AKC), or Abnormal 2 (older AKC). The abnormal ISC states in the crypt structures are distinct from the cancer stem cells found in the cancer group. Trajectory analysis revealed that each stem cell population follows a distinct differentiation sequela, with abnormal ISC giving rise to transcriptionally divergent epithelial lineages. This was validated by integrating scRNA-seq data with spatial transcriptomics data from the same lines, showing that abnormal epithelial populations progressively overtake the normal crypt epithelium while retaining crypt structure. Differential gene expression analysis and Gene Set Enrichment Analysis showed that normal ISC was enriched for classic stemness markers (e.g., Lgr5, Lrig1, Smoc2). Abnormal 1 ISC began to lose these features and instead exhibited increased expression of cell-cycle regulators (e.g., Ccna2, Aurkb) and markers of oxidative stress (e.g., Prdx4, Gsta3, Gpx2). Gene expression profiles in Abnormal 2 ISC showed enrichment of pathways controlling p53 (e.g., Cdkn1a, Smad7), RAS/MAPK (e.g., Map2k1, Epha2), and PI3K/Akt/mTOR signaling (e.g., Akt3, Pik3r3, Mtor), as well as for an inflammatory response (e.g., Fos/Jun, Nfkbiz, Il18). Collectively, these findings show that early, non-transforming mutations push ISC into preneoplastic states marked by increased proliferation and stress response signaling favorable for malignant progression. Citation Format: Yujin Lee, Moray J. Campbell, James C. Fleet. Single-cell mapping of the colon epithelium reveals a pre-malignant stem cell that defines early transformation risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5922.

Cannabidiol (CBD) effects on bone metabolism in postmenopausal osteoporosis remain unclear. While endocannabinoids and phytocannabinoids bind to receptors in bone cells, direct evidence of CBD's bone-protective effects is lacking. We evaluated the effects of CBD on bone metabolism in ovariectomized (OVX) rat model of estrogen deficiency. Twelve-week study with treatment initiated 2 wk after the surgery was conducted. Five experimental groups were established: sham-operated with vehicle (SHM/VEH), sham with CBD (SHM/CBD5), OVX with vehicle (OVX/VEH), OVX with 17β-estradiol (OVX/E2), and OVX with CBD (OVX/CBD5). Cannabidiol was administered at 5 mg/kg/d via osmotic pumps. Micro-CT of the distal femur revealed that trabecular bone mass in OVX/CBD5 decreased similarly to OVX/VEH, indicating no protective effect. Serum bone turnover markers showed increased bone resorption in OVX/CBD5 compared to OVX/VEH. Gene expression analysis revealed that estrogen significantly reduced Ctsk gene expression compared to OVX/VEH, while CBD showed no significant differences. No significant changes were observed in cannabinoid receptor expression or bone metabolism in sham-operated rats receiving CBD. While CBD (5 mg/kg/d) was well-tolerated, it did not mitigate OVX-induced bone loss in skeletally mature rats. Consequently, CBD should not be considered a monotherapy for postmenopausal osteoporosis, though it appears safe for other potential medical applications.

A challenge for public health is to make recommendations for the intake of vitamin D to optimize health and prevent disease. Vitamin D supplementation can be accomplished using vitamin D2 (from fungi), vitamin D3 (from vertebrate sources), or as the prehormone 25 hydroxyvitamin D3 (25OHD3). While these three sources are generally effective for raising and maintaining vitamin D status, their effect is not identical. This review will summarize the differences between these molecules in their routes of absorption, their metabolism, degradation, and molecular function.

IntroductionThe intestine has molecular and functional diversity across the proximal-distal and the crypt-villus axes, so it is imperative to determine the common and compartment-specific molecular actions of vitamin D. However, very little work on vitamin D mediated gene regulation has been done in normal human intestine. Here, we examined the impact of 1,25-dihydroxyvitamin D (1,25(OH)2D3) on cultures of human intestinal epithelium derived from duodenum (Dd) and distal colon (Co) biopsies of 6 subjects per tissue.MethodsHuman enteroids and colonoids were cultured for 3 days to promote a stem cell phenotype (undifferentiated, Un) or to induce differentiation (Diff) and then treated with vehicle control or 1,25(OH)2D3 (100 nM). 24h following treatment enteroids/colonoids were collected, RNA was isolated and RNA-seq was performed using paired-end Illumina sequencing (analysis in R using DESeq2).Results and discussionRNA-seq analysis showed that VDR mRNA is present in all four cultures tested (DdUn, DdDiff, CoUn, CoDiff) and it is not altered by 1,25(OH)2D3 treatment, intestinal segment, or differentiation status. 1,25(OH)2D3 induced the classic intestinal target genes TRPV6, ATP2B1 and CYP24A1 in all four culture groups while S100G was induced only in DdDiff. While 63 genes were vitamin D regulated across all four cultures (55 up, 8 down), we found that vitamin D regulated subgroups of genes within Dd, Co, Un, or Diff groups as well as set of genes that were unique to each culture. Functional analysis revealed several vitamin D-enriched gene ontologies or pathways including those for xenobiotic/drug metabolism in all four cultures. In differentiated cultures vitamin D induced genes were enriched for functions like regulation of barrier function through regulation of Rho GTPases and metabolism of lipids while vitamin D downregulated genes in Un groups were enriched for activities like water transport. These results provide new insight into 1,25(OH)2D3 genomic action in the functionally distinct compartments and segments of human intestine and suggest multiple regulatory effects of vitamin D in human intestinal physiology.

Early onset colorectal cancer (EOCRC) is increasing yet the mechanism for the accelerated shift to a younger age is unknown. We hypothesize that sporadic gene mutations that are necessary for colon carcinogenesis but not sufficient to cause cancer, create a preneoplastic cell state for increased risk of transformation. To identify transitional cell phenotypes, we used Spatial Transcriptomics (ST) on distal colon samples from Car1-Cre (CAC) mice with Cre expression limited to ∼6% of the distal colon/rectum epithelium, where EOCRC is more prevalent. CAC;ROSAmT/mG (CR) mice (where eGFP marks cells expressing Cre) were crossed to those with floxed oncogene alleles to generate mice with one (Apc+/-, ACR) or two (Apc+/-;KrasG12D/wt, AKCR) non-transforming mutations. ACR mice had normal crypt morphology and low adenoma incidence (<5% of mice), while 4-week-old AKCR mice exhibited crypt elongation, and 8-10 wk-old AKCR developed 3-8 advanced adenomas/mouse. Distal colon Swiss rolls (n=9) and tumors (n=3) with phenotypes ranging from normal to malignant were collected and processed using the 10X Visium Spatial Gene Expression System. Sequencing data was processed, and sample data were pooled for analysis using Seurat. Spatial counts were normalized, dimensionality was reduced with PCA, then clustered and visualized as UMAP projections. Clusters were annotated to known cell types, and differential gene expression (DEG) analysis was followed by functional annotation using GO and Pathway terms. In normal tissue, clusters for the crypt base, middle, and top were identified. These spatially restricted clusters were replaced in early AKCR crypts and again in late AKCR crypts. The clusters from late AKCR crypts were distinct from those in ACR tumors, which were different from clusters in AKCR tumors. This indicates the existence of transition states from normal to “not normal, not cancer” to early adenoma to late adenoma. Normal epithelial clusters were enriched for genes controlling typical colonic functions, e.g. metabolism, and transmembrane transport pathways. AKCR crypt clusters lost these functions and gained others, e.g. immune and antimicrobial genes normally associated with Paneth cells (e.g., Defa22, Defa24, Defa30), gene markers associated with CRC tumors (e.g. Egfr, Hamp, Plet1, Wfdc18), genes for xenobiotic inactivation (Maoa, various Cyp, Gst, Ugt, and Sult genes). Compared to late AKCR crypts, AKCR tumor clusters lost even more colonic functions (e.g. downregulation of differentiation marker Krt20 and xenobiotic metabolism markers) and expressed genes related to mitosis, DNA replication, and DNA repair, highlighting a transition to uncontrolled proliferation and genomic instability. Our findings delineate key early molecular transitions in epithelial cells during colon carcinogenesis that may increase the risk of transformation. Yujin Lee, James C. Fleet. Spatial transcriptomic profiling of the mouse distal colon during early carcinogenesis reveals transitional cell states that precede cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6602.

Objectives: Accumulating clinical evidence from experimental and observational studies with humans suggests that edible mushrooms may have beneficial effects on markers of brain health. This study examined the effects of daily consumption of fresh Agaricus bisporus (cremini mushrooms) exposed to ultraviolet (UV) light on indices of anxiety, depression, mood, cognitive function, and well-being in middle-aged and older adults. Methods: Over a 6-week period, adults (n = 41 (19 M/22 F), age 43 ± 11 y; BMI 29.8 ± 5.9 kg/m2, mean ± SD) without severe depression, cardiovascular disease, or Type 2 Diabetes consumed two daily servings (168 g/d wet weight) of cremini mushrooms intended to provide 400 IU/serving (800 IU/d) of vitamin D2 (n = 20) or 2 tsp/d of breadcrumbs (control, n = 21). Assessments conducted at baseline and week 6 included General Anxiety Disorder-7 (GAD-7), Beck’s Depression Inventory (BDI-II), Patient Health Questionnaire (PHQ-9), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Profile of Mood States (POMS), and Medical Outcomes Study 36-Item Short-Form Health Survey Version 2 (SF36v2). Results: Consuming UV light-exposed mushrooms did not improve brain health outcomes. Independent of mushroom consumption, over time, there were improvements in immediate memory (RBANS), language (RBANS), and depression (BDI-II and PHQ-9). Conclusions: There were no differences observed between groups in the investigated indices of brain health. However, improvements over time were observed in Beck’s Depression Inventory and the Immediate Memory and Language domains in the RBANS, independent of mushroom consumption. Overall, consuming 2 servings/d of UV-exposed mushrooms for six weeks may not improve indices of brain health.

Vitamin D insufficiency (VDI) is primarily determined by serum levels of calcidiol, which serves as a biomarker for the less abundant but most potent bioactive metabolite, calcitriol. However, population studies often show discordance between calcidiol and calcitriol. Here, a genetically diverse population of 7 inbred mouse strains was used to investigate the role of interindividual genetic differences in driving calcidiol-to-calcitriol discordance under vitamin D sufficient (VDS) vs depleted (VDD) conditions. We found high interstrain variability in calcitriol that was discordant with calcidiol under VDS and VDD conditions. However, under VDS conditions, stratification by calcitriol level revealed that strains with serum calcitriol >60 pM (HighC) exhibited the expected positive calcidiol-to-calcitriol association, whereas strains with low calcitriol (<60 pM, LowC) did not. Thus, discordance under VDS was driven by genetically divergent strains with LowC. Discordance under VDD was not associated with LowC. LowC was not caused by increased calcitriol degradation or by transcriptional dysregulation of canonical vitamin D metabolism enzymes. Instead, LowC strains exhibited low renal expression of Lrp2 (megalin), the primary transporter required for renal calcitriol production. LowC strains also exhibited reduced renal expression of the vitamin D receptor (Vdr) and several target genes, demonstrating impaired vitamin D signaling. These findings reveal novel, naturally occurring genetic determinants of VDI that function by disrupting calcitriol production and signaling in a manner that cannot be predicted by calcidiol levels. Cross-species conservation of this phenomenon would have important implications for clinical management of VDI and related disease risks across genetically diverse populations.

Preterm infants born to mothers with preeclampsia, a disease of vascular dysfunction, are at increased risk for bronchopulmonary dysplasia (BPD). Endothelial cells are critical in both maintaining proper vascular function and coordinating lung development. Understanding the mechanisms contributing to BPD in the setting of preeclampsia and how preeclampsia impacts pulmonary endothelial cells (PECs) in the newborn lung are required to decrease the burden of BPD. Vitamin D has been shown to improve lung angiogenesis and lung development in inflammatory models of BPD, but its therapeutic potential in the setting of preeclampsia is unknown. We hypothesized that intraamniotic (IA) treatment with the biologically active form of vitamin D, 1,25 dihydroxyvitamin D [1,25(OH)2D], will preserve lung growth in an experimental model of BPD induced by antenatal exposure to soluble vascular endothelial growth factor receptor-1 [sFlt-1 (soluble fms-like tyrosine kinase 1)]. Fetal rats were exposed to saline (control), sFlt-1 alone, 1,25(OH)2D alone, or simultaneous sFlt-1 + 1,25(OH)2D via IA injection during the late canalicular stage of lung development and delivered 2 days later. IA treatment with 1,25(OH)2D in sFlt-1-exposed pups improved lung alveolar and vascular growth and function at 14 days of life. PECs orchestrate alveolar development, and we demonstrate that IA sFlt-1 exposure alone decreased in vitro growth and tube formation of PECs isolated from newborn pups and that PECs from pups coexposed to IA sFlt-1 and 1,25(OH)2D demonstrated increased growth and tube formation. We conclude that IA 1,25(OH)2D treatment improves distal lung development during sFlt-1 exposure through preservation of angiogenesis in the developing lung.NEW & NOTEWORTHY This study highlights that experimental BPD induced by intraamniotic sFlt-1 is associated with impaired growth in postnatal pulmonary endothelial cells. We demonstrate that 1,25(OH)2D may be a therapeutic option to improve lung development through enhancement of VEGF signaling and preservation of early pulmonary endothelial growth in the newborn rat lung.

Expression of 25 hydroxyvitamin D 24 hydroxylase from the Cyp24a1 gene mediates 1,25 dihydroxyvitamin D (1,25(OH)2D) catabolism but gaps exist in our understanding of this enzyme's physiologic importance. Here, we used tamoxifen to induce Cyp24a1 gene knockout (KO) in adult mice (50 mg Tamoxifen /g BW, ip, 5 d, at 11 wks of age) or intestinal-epithelial-cell-specific knock-out mice (IEC KO) to evaluate the role of CYP24A1 in adult mice and the contribution of the intestine to vitamin D (Vit D) metabolism. At 12-wks mice were euthanized and serum was analyzed for Vit D metabolites by LC MS/MS while duodenal (Dd) and kidney (Kd) mRNA levels were quantified using qPCR. Adult Cyp24a1 KO mice had higher 25 hydroxyvitamin D (25(OH)D, + 185 %) and 1,25(OH)2D (+41.4 %) levels and reduced levels of 1,24,25(OH)3D (-53.7 %). No changes in serum Vit D metabolites were seen in adult mice lacking one Cyp24a1 allele (HT). In kidney, compensatory changes in Cyp27b1 mRNA (-85.3 % in KO, -36.5 % in HT) and Cyp24a1 mRNA (+147 % in KO, +43 % in HT) were observed. No changes in Dd Trpv6 or S100g mRNA were observed and Dd Cyp3a13 mRNA did not compensate for Cyp24a1 gene loss. Neither serum Vit D metabolites nor Dd Trpv6 and S100g mRNA were changed in IEC KO mice but there was a trend towards elevated renal Cyp24a1 mRNA (+61 %, p = 0.06). Our data in adult KO mice indicate that CYP24A1 has an important physiologic impact on Vit D metabolism while IEC KO data suggests that local degradation of the hormone by CYP24A1 is not a strong regulator of intestinal Vit D action or systemic vitamin D metabolism.

Environmental factors and genetic variation individually impact bone. However, it is not clear how these factors interact to influence peak bone mass accrual. Here we tested whether genetically programmed high bone formation driven by missense mutations in the Lrp5 gene (Lrp5A214V) altered the sensitivity of mice to an environment of inadequate dietary calcium (Ca) intake. Weanling male Lrp5A214V mice and wildtype littermates (control) were fed AIN-93G diets with 0.125%, 0.25%, 0.5% (reference, basal), or 1% Ca from weaning until 12 weeks of age (ie, during bone growth). Urinary Ca, serum Ca, Ca regulatory hormones (PTH, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)), bone parameters (μCT, ash), and renal/intestinal gene expression were analyzed. As expected, low dietary Ca intake negatively impacted bones and Lrp5A214V mice had higher bone mass and ash content. Although bones of Lrp5A214V mice have more matrix to mineralize, their bones were not more susceptible to low dietary Ca intake. In control mice, low dietary Ca intake exerted expected effects on serum Ca (decreased), PTH (increased), and 1,25(OH)2D3 (increased) as well as their downstream actions (ie, reducing urinary Ca, increasing markers of intestinal Ca absorption). In contrast, Lrp5A214V mice had elevated serum Ca with a normal PTH response but a blunted 1,25(OH)2D3 response to low dietary Ca that was reflected in the renal 1,25(OH)2D3 producing/degrading enzymes, Cyp27b1 and Cyp24a1. Despite elevated serum Ca in Lrp5A214V mice, urinary Ca was not elevated. Despite an abnormal serum 1,25(OH)2D3 response to low dietary Ca, intestinal markers of Ca absorption (Trpv6, S100g mRNA) were elevated in Lrp5A214V mice and responded to low Ca intake. Collectively, our data indicate that the Lrp5A214V mutation induces changes in Ca homeostasis that permit mice to retain more Ca and support their high bone mass phenotype.

Abstract We tested whether lifelong modification of vitamin D signaling can alter the progression of early prostate carcinogenesis in studies using mice that develop high-grade prostatic intraepithelial neoplasia that is similar to humans. Two tissue-limited models showed that prostate vitamin D receptor (VDR) loss increased prostate carcinogenesis. In another study, we fed diets with three vitamin D3 levels (inadequate = 25 IU/kg diet, adequate for bone health = 150 IU/kg, or high = 1,000 IU/kg) and two calcium levels (adequate for bone health = 0.5% and high = 1.5%). Dietary vitamin D caused a dose-dependent increase in serum 25-hydroxyvitamin D levels and a reduction in the percentage of mice with adenocarcinoma but did not improve bone mass. In contrast, high calcium suppressed serum 1,25-dihydroxyvitamin D levels and improved bone mass but increased the incidence of adenocarcinoma. Analysis of the VDR cistrome in RWPE1 prostate epithelial cells revealed vitamin D–mediated regulation of multiple cancer-relevant pathways. Our data support the hypothesis that the loss of vitamin D signaling accelerates the early stages of prostate carcinogenesis, and our results suggest that different dietary requirements may be needed to support prostate health or maximize bone mass. Significance: This work shows that disrupting vitamin D signaling through diet or genetic deletion increases early prostate carcinogenesis through multiple pathways. Higher-diet vitamin D levels are needed for cancer than bone.

Supplementary Table Legends 1-7, Figure Legends 1-9 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D3 in Prostate Progenitor/Stem Cells

1,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3)-mediated intestinal calcium (Ca) absorption supplies Ca for proper bone mineralization during growth. We tested whether vitamin D receptor (VDR)-mediated 1,25(OH)(2)D-3 signaling is critical for adult Ca absorption and bone by using mice with inducible Vdr gene knockout in the whole intestine (villin-CreER(T2+/-) x Vdr(f/f), WIK) or in the large intestine (Cdx2-CreER(T2+/-) xVdr(f/f), LIK). At 4-month-old, Vdr alleles were recombined (0.05 mg tamoxifen/g BW, intraperitoneally [i.p.], 5 days) and mice were fed diets with either 0.5% (adequate) or 0.2% (low) Ca. Ca absorption was examined after 2 weeks while serum 1,25(OH)(2)D-3, bone mass, and bone microarchitecture were examined after 16 weeks. Intestinal and renal gene expression was measured at both time points (n = 12/genotype/diet/time point). On the 0.5% Ca diet, all phenotypes in WIK and LIK mice were similar to the controls. Control mice adapted to the 0.2% low-Ca diet by increasing renal Cyp27b1 mRNA (3-fold), serum 1,25(OH)(2)D-3 level (1.9-fold), and Ca absorption in the duodenum (Dd, + 131%) and proximal colon (PCo, + 28.9%), which prevented bone loss. In WIK mice, low-Ca diet increased serum 1,25(OH)(2)D-3 (4.4-fold) but Ca absorption remained unaltered in the Dd and PCo. Consequently, significant bone loss occurred in WIK mice (e.g., cortical thickness, Ct.Th, -33.7%). LIK mice adapted to the low-Ca diet in the Dd but not the PCo, and the effect on bone phenotypes was milder (e.g., Ct.Th, -13.1%). Our data suggest intestinal VDR in adult mice prevents bone loss under low Ca intake but is dispensable under adequate calcium intake.