Disease burden measured by patient-reported outcomes: does psoriatic arthritis feel worse than rheumatoid arthritis? A cross-sectional nationwide study.

OBJECTIVES: To study the subjective disease burden of patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA), using patient-reported outcomes (PROs) cross-sectionally. METHODS: Data of 3598 patients with PsA and 13913 with RA were extracted from the database. Measures included the VAS-values of pain, fatigue and patient global assessment (PGA), HAQ, and disease activity at the most recent visit/remote contact in the period 1.2020 to 9.2021. Values were compared between patients with PsA and RA overall, and by sex and age (<50, 50-59, 60-69 and ≥70 years). Regression analyses were applied. RESULTS: The overall median (IQR)-values for pain were 29 (10, 56) for PsA and 26 (10, 51) for RA, 29 (9, 60) and 28 (8, 54) for fatigue, 28 (10, 52) and 29 (11, 51) for PGA, 0.4 (0, 0.9) and 0.5 (0, 1.0) for HAQ (p<0.001 for all comparisons; adjusted for sex and age). The median (IQR)-values for pain, fatigue, PGA and HAQ were higher for PsA vs. RA in most age groups for males and females. All PROs were higher in older patients with both diagnoses. The median values for DAS28, doctor global assessment, ESR and CRP were 1.9 vs. 2.0, 8 vs. 8, 7 vs. 8 and 2 vs. 3 in PsA and RA, respectively. CONCLUSIONS: Overall, both PsA and RA groups showed moderate disease control by patients' perspective, but the burden of disease was higher especially in women with PsA compared to RA. Disease activity was similar and low in both diseases.

Incidence of seropositive rheumatoid arthritis in population-based studies in Northern Savo, Finland, during 1980-2020.

The objective is to evaluate the incidence of seropositive rheumatoid arthritis (RA) over 40-year period in Northern Savo, Finland. Data on new seropositive RA patients according to the American College of Rheumatology (ACR) 1987 classification criteria were collected in 2020-2021. In 2020 data on tobacco exposure, patient-reported dental health and living in residences with fluoridated tap water were gathered. Incidence rates were estimated and age- and gender-adjusted to Northern Savo population. The results were compared with data acquired in studies from 1980, 1990, 2000, and 2010. In 2020, 46 seropositive RA patients (21 females and 25 males) were recorded. The crude incidence of seropositive RA fulfilling the ACR 87 criteria in 2020 was 22.3 (95% CI 16.3 to 29.8)/100 000 and age and gender-adjusted 22.3 (95% CI 15.9 to 28.8)/100 000. Tobacco exposure > 5 pack years occurred 18% of females and 56% of males. Only 16% of males were full dentate. A total of 242 incident seropositive RA (age ≥ 16 years, 55% females) were identified in all study years. No differences in the gender-specific incidence rates in each cohort or in incidence between the studies every 10 years were recorded. The incidence of seropositive RA decreased in the age group < 55 years, p = 0.003. There was no change in the incidence of seropositive RA between genders or the study years. A declining trend for occurrence of seropositive RA in the young and early middle-aged population may reflect change in risk factors.

Preliminary Report: Osteoarthritis and Rheumatoid Arthritis Synovial Fluid Increased Osteoclastogenesis In Vitro by Monocyte Differentiation Pathway Regulating Cytokines.

Background: Rheumatoid arthritis (RA) and osteoarthritis (OA) are common joint diseases associated with changes in local, as well as systemic bone structure and osteoclast function. We investigated how the different soluble inflammatory stimuli in these diseases can affect osteoclastogenesis and bone resorption in vitro. Methods. Human peripheral blood mononuclear cell-derived osteoclasts were cultured on bone slices with serum from treatment-naïve RA patients and healthy controls and with synovial fluid samples acquired from RA and OA patients. The concentrations of 29 different cytokines and related proteins, including RANKL and OPG, were analyzed in the fluids tested. Results: RA serum and synovial fluid increased both osteoclastogenesis and bone resorption. Osteoclastogenesis and activity increased more in the cultures containing OA than RA synovial fluid. The osteoclasts cultured in different culture media exhibited different phenotypes, especially the cells cultured with OA synovial fluid were generally larger and had more nuclei. A general increase in proinflammatory cytokines in RA synovial fluid and serum was found. Surprisingly, OA synovial fluid showed lower levels of osteoclastogenesis inhibiting cytokines, such as IL-4 and IL-10, than RA synovial fluid, which at least partly explains more pronounced osteoclastogenesis. No significant difference was found in RANKL or OPG levels. Conclusion: The proinflammatory stimulus in OA and RA drives the monocyte differentiation towards inflammatory osteoclastogenesis and altered osteoclast phenotype.

Similar levels of disease activity and remission rates in patients with psoriatic arthritis and rheumatoid arthritis-results from the Finnish quality register.

OBJECTIVES: To compare the current disease activity and remission rates, and their regional variation in patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA) in Finland. METHODS: Data of patients' most recent visit in 1/2020-9/2021 were extracted from the Finnish Rheumatology Quality Register. Measures for disease activity and remission included joint counts, DAS28, cDAPSA, CDAI, the Boolean definition, and physician assessment. Regression analyses were applied, adjusted for age and sex. RESULTS: Data of 3598 patients with PsA (51% female, mean age 54 years) and 13,913 patients with RA (72% female, 74% ACPA-positive, mean age 62 years) were included. The median (IQR) DAS28 was 1.9 (1.4, 2.6) in PsA and 2.0 (1.6, 2.7) in RA (p = 0.94); for cDAPSA, the median (IQR) values were 7.7 (3.1, 14) in PsA and 7.7 (3.3, 14) in RA (p < 0.001). In all regions in both diseases, the median DAS28 was ≤ 2.6 and the median cDAPSA < 13. Remission rates included DAS28 < 2.6 in 73% in PsA and 69% in RA (p = 0.17) and Boolean remission in 17% in PsA and 15% in RA (p < 0.001). By other definitions of remission, the rates ranged between 30% and 46%. Methotrexate was currently used by 49% in PsA and 57% in RA (p < 0.001). Self-administered bDMARDs were currently used by 37% in PsA and 21% in RA (p < 0.001). CONCLUSION: The overall disease activity was low and similar in patients with PsA and RA across the country. Remission rates varied between 15 and 73%, depending on the definition but were similar in PsA and RA. Key Points • The disease activity and clinical picture was similar between patients with PsA and RA, in a cross-sectional setting in 1.2020-9.2021. • A significant majority of patients with PsA had low disease activity or were in remission according to cDAPSA. Majority of patients with RA were in remission according to DAS28. • Patients with PsA and RA used methotrexate similarly. The utilization of bDMARDs was more prevalent in patients with PsA.

Intense symptoms of pain are associated with poor sleep, fibromyalgia, depression and sleep apnea in patients with rheumatoid arthritis and psoriatic arthritis. A register-based study.

OBJECTIVES: To study whether poor sleep and comorbidities are associated with high symptom levels of patient-reported outcomes (PROs) pain, patient global assessment and fatigue in patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA), in a nation-wide cross-sectional setting. METHODS: Clinical data were extracted from The Finnish Rheumatology Quality Register between 1.2021 and 9.2022. Self-reported sleep was categorized as "good" (little/no difficulties) or "poor" (great difficulties/can't) sleep. Data concerning comorbidities were collected from national registers. Descriptive statistics were used. Regression analyses were applied to analyze independent associations of sleep status, comorbidities and disease activity with pain in RA and PsA, adjusting for age and sex. RESULTS: Among 13,512 patients with RA, 6052 [mean (SD) age 62 (13), 71% female] had sleep status reported; in PsA 1861/3636 [age 55 (13), 48% female]. In RA, 5072 (84%) reported good and 980 (16%) poor sleep; the corresponding numbers in PsA were 1460 (78%) and 401 (22%). Median values for objective disease activity were low and similar in patients with poor sleep and good sleep in both diseases. Among patients with no swollen joints, the median values for PROs were approximately three times higher for patients with poor sleep vs. good sleep in both diagnoses (P<0.001). In regression analyses, "poor" sleep was independently associated with higher symptoms in pain [B (95%CI) 20 (18,22) in RA and 23 (19, 26) in PsA], followed by comorbid fibromyalgia, as well as depression in RA and sleep apnea in PsA. CONCLUSION: "Poor" sleep quality and comorbidities are independently associated with pain. Patient's sleep status is important to know especially in patients with severe symptoms without objective disease activity.

1,25(OH)2D3 and its analogue calcipotriol inhibit the migration of human synovial and mesenchymal stromal cells in a wound healing model - A comparison with glucocorticoids.

Vitamin D analogue calcipotriol is currently used in the local treatment of psoriasis. However, it also has antiproliferative and anti-inflammatory effects in the cells of the joint - suggesting a possible benefit in local treatment of arthritis. In this study, calcipotriol was studied in different in vitro methods to find out its effect on synovial and mesenchymal stromal cells. Primary human cell lines of osteoarthritis or rheumatoid arthritis patients (five mesenchymal stromal cells, MSC, and four synovial stromal cells, SSC) were cultured to study migration and proliferation of the cells in a wound healing model. The media was supplemented with calcipotriol, 1,25(OH)2D3, dexamethasone, betamethasone, methylprednisolone or control solution in 1-100 nM concentrations. To see possible toxic effects of calcipotriol, concentrations up to 10 µM in SSCs and MSCs were studied in apoptosis and necrosis assays in four cell lines. Calcipotriol and 1,25(OH)2D3, as well as the three glucocorticoids, reduced the migration of both SSCs and MSCs. In SSCs, the effect of calcipotriol and 1,25(OH)2D3 was at least as effective as with glucocorticoids, while with MSCs, the glucocorticoids were stronger inhibitors of migration. The antimigratory of calcipotriol and 1,25(OH)2D3 was consistently maintained in 10 µM and 1 µM. Calcipotriol was not toxic to MSCs and SSCs up to concentrations of 10 µM. Calcipotriol, as well as 1,25(OH)2D3, exerts antimigratory and antiproliferative effects on human SSCs and MSCs of the joint. These effects are not caused by apoptosis or necrosis. Both calcipotriol and 1,25(OH)2D3 have similar effects as glucocorticoids without apparent toxicity, suggesting that calcipotriol might be an eligible candidate to the local treatment of arthritis with a broad therapeutic window.

Initial presentation of early rheumatoid arthritis.

OBJECTIVES: To study the joint distribution and clinical picture of rheumatoid arthritis (RA) at the initial presentation in seropositive (anti-citrullinated protein antibody (ACPA) and/or rheumatoid factor (RF) positive) and negative patients and the effect of duration of symptoms on the clinical picture. METHODS: Data of patients who received reimbursement for DMARDs for newly diagnosed RA in 1/2019 to 9/2021 were extracted from the national databases. Joint counts, presence of symmetrical swelling, other disease activity measures, and patient reported outcomes (PROs) were compared in seropositive and negative patients. Regression analyses were applied to compare clinical variables in patients with duration of symptoms of <3, 3-6, and >6 months, adjusted for age, sex, and seropositivity. RESULTS: Data of 1816 ACPA and RF-tested patients were included. Symmetrical swelling was present in 75% of patients. Seronegative versus positive patients had higher value for all disease activity measures and PROs including median swollen joint count (SJC46 10 versus 5) and DAS28 (4.7 versus 3.7), (p<0.001). Patients diagnosed in <3 months had higher median pain VAS (62 versus 52 and 50, p<0.001) and HAQ (1.1 versus 0.9 and 0.75, p = 0.002) compared to those with a duration of symptoms of 3-6 and >6 months. Patients diagnosed >6 months were ACPA-positive more frequently (77% versus 70% in other groups, p = 0.045). CONCLUSION: Incident RA presents mainly as symmetric arthritis. Seronegative patients have higher disease burden at the initial presentation. Patients experiencing more severe pain and decreased functional ability are diagnosed earlier, regardless of ACPA- status.

No adverse effects on periarticular tissue by intra-articular vitamin D analogue calcipotriol in a reduced-dose zymosan-induced arthritis model in rats.

Calcipotriol, a vitamin D analogue, is an antiproliferative and anti-inflammatory drug currently used in psoriasis. Here, our aim was to analyse the safety of calcipotriol for cartilage and bone in alleviated-dose (0.1 mg instead of usual ≥1mg dose) zymosan-induced arthritis in rats. Theoretically, high doses of vitamin D or analogues could have detrimental effects on bone or cartilage. The rats were divided into four groups: vehicle (n = 9), dexamethasone 0.1 mg/kg (n = 9), calcipotriol 0.1 mg/kg (n = 8) and negative control (n = 10) with no injections. Arthritic rats were given phosphate-buffered saline (PBS) injections to left knees as a control. After euthanasia on day 8, all knees were imaged with micro-computed tomography for surface lesions and decalcified for histological analyses. Contrary to our expectations, no significant changes could be observed in the tomography data and histological scores among the three treatment groups or between the vehicle-treated and non-arthritic group. Calcipotriol did not cause adverse effects on cartilage or subchondral bone within a week, suggesting that it could be safely used in local treatment of arthritis. The alleviated model caused synovitis with local and systemic inflammatory response without cartilage erosions, which might be useful in studying self-limiting synovitis where cartilage or bone effects are not of primary interest.

A single intra-articular dose of vitamin D analog calcipotriol alleviates synovitis without adverse effects in rats.

1,25-dihydroxyvitamin-D3 and its derivatives have shown anti-arthritic and chondroprotective effects in experimental animal models with prophylactic dosing. The purpose of this preliminary study was to test the efficacy and safety of calcipotriol, vitamin D analog, as a treatment for a fully-developed knee arthritis in Zymosan-induced arthritis (ZIA) model. Forty 5-month-old male Sprague-Dawley rats were randomized into three arthritis groups and a non-arthritic control group with no injections (10 rats/group). A day after Zymosan (0.1 mg) had been administrated into the right knee joints, the same knees were injected with calcipotriol (0.1 mg/kg), dexamethasone (0.1 mg/kg) or vehicle in a 100 µl volume. The left control knees were injected with saline (PBS) on two consecutive days. All injections, blood sampling and measurements were performed under general anesthesia on days 0, 1, 3 and 8. Internal organs and knees were harvested on day 8 and the histology of the whole knees was assessed blinded. Joints treated with calcipotriol showed a milder histological synovitis than those treated with vehicle (p = 0.041), but there was no statistically significant difference between the dexamethasone and vehicle groups. The clinical severity of arthritis did not differ between the arthritis groups measured by body temperature, swelling of the knee, thermal imaging, clinical scoring or cytokine levels on days 1, 3 and 8. Weight loss was bigger in rats treated with dexamethasone, propably due to loss of appetite,compared to other arthritis groups on days 2-3 (p<0.05). Study drugs did not influence serum calcium ion and glucose levels. Taken together, this preliminary study shows that a single intra-articular injection of calcipotriol reduces histological grade of synovitis a week after the local injection, but dexamethasone did not differ from the vehicle. Calcipotriol may have an early disease-modifying effect in the rat ZIA model without obvious side effects.

Differential synovial tissue expression of TLRs in seropositive and seronegative rheumatoid arthritis: A preliminary report.

Toll-like receptors (TLRs) are known to have an important role in triggering the innate immune response and in priming antigen-specific adaptive immunity and inflammation. The differences in synovial tissue expression of the TLRs between seronegative and seropositive rheumatoid arthritis (RA) were examined from 9 seropositive RA, 5 seronegative RA and 4 osteoarthritis (OA) patients. Synovitis status was assessed using Krenn's scoring and TLR 1-9 expression by immunohistochemistry. Tissue citrulline content was analysed by HPLC method. In RA TLR expression was generally higher than in OA. TLR2 expression was higher in both seronegative and seropositive RA compared to OA. TLR 1, 4 and 8 expressions were higher in seropositive RA than in seronegative RA or in OA. For TLRs 3, 5, 6, 7 and 9 local differences of expression were found between groups. TLR 1-9 expression correlated with the synovitis grade. No statistical difference was found in synovial tissue citrulline content between the groups. In seropositive RA, the TLR repertoire in the synovial tissue differs from seronegative RA and could explain differences in disease outcomes. The high expression of protein sensing (TLR1, TLR2 and TLR4) and nucleic acid sensing TLRs (TLR7, TLR8 and TLR9) in the seropositive RA could make the synovium primed for reacting to citrullinated proteins and nucleic acids that could be released to extracellular space in formation of neutrophil extracellular traps. This reactivity could be augmented by Fc receptor activation by anti-citrullinated protein antibody immunocomplexes associated with seropositive RA.

Distinct fatty acid signatures in infrapatellar fat pad and synovial fluid of patients with osteoarthritis versus rheumatoid arthritis.

BACKGROUND: Infrapatellar fat pad (IFP) has recently emerged as a potential source of inflammation in knee arthropathies. It has been proposed to be one source of adipocytokines, fatty acids (FA), and FA-derived lipid mediators that could contribute to the pathophysiological processes in the knee joint. Alterations in synovial fluid (SF) lipid composition have been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA). The aim of the present study was to compare the FA signatures in the IFP and SF of RA and OA patients. METHODS: Pairs of IFP and SF samples were collected from the same knees of RA (n = 10) and OA patients (n = 10) undergoing total joint replacement surgery. Control SF samples (n = 6) were harvested during diagnostic or therapeutic arthroscopic knee surgery unrelated to RA or OA. The FA composition in the total lipids of IFP and SF was determined by gas chromatography with flame ionization and mass spectrometric detection. RESULTS: Arthropathies resulted in a significant reduction in the SF proportions of n-6 polyunsaturated FA (PUFA), more pronouncedly in OA than in RA. OA was also characterized with reduced percentages of 22:6n-3 and lower product/precursor ratios of n-3 PUFA. The proportions of total monounsaturated FA increased in both RA and OA SF. Regarding IFP, RA patients had lower proportions of 20:4n-6, total n-6 PUFA, and 22:6n-3, as well as lower product/precursor ratios of n-3 PUFA compared to OA patients. The average chain length of SF FA decreased in both diagnoses and the double bond index in OA. CONCLUSIONS: The observed complex alterations in the FA signatures could have both contributed to but also limited the inflammatory processes and cartilage destruction in the RA and OA knees.

Pharmacokinetics of intra-articular vitamin D analogue calcipotriol in sheep and metabolism in human synovial and mesenchymal stromal cells.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC-MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Cytokine data obtained from synovial stromal cells of patients with rheumatoid arthritis or osteoarthritis.

In this article, we share the raw cytokine data obtained from basal and stimulated synovial stromal cells cultured from patients with rheumatoid arthritis or osteoarthritis. This data article is related to the research article entitled "1,25D3 and calcipotriol, its hypocalcemic analog, exert a long-lasting anti-inflammatory and anti-proliferative effect in synoviocytes cultured from patients with rheumatoid arthritis and osteoarthritis (1). Cytokine levels were analyzed by a magnetic bead-based multiplex assay (a panel of 27 important cytokines) in two separate sets of experiments. The first was conducted with IL-1ß and 1,25(OH)2D3 and the other with TNFα, calcipotriol, i.e. the hypocalcemic analog 1,25(OH)2D3, and dexamethasone. The raw data of this article display the individual variation in basal secretion of cytokines and in their response to different stimuli.

1,25(OH)2D3 and calcipotriol, its hypocalcemic analog, exert a long-lasting anti-inflammatory and anti-proliferative effect in synoviocytes cultured from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES: We investigated the effects of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), i.e. biologically active vitamin D and calcipotriol, a vitamin D analog, on growth and secretion of inflammatory mediators in synovial stromal cells (SSC) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Synovial stromal cells (SSC) isolated during knee prosthesis surgery from four patients with RA and four with OA were exposed to 1,25(OH)2D3 or calcipotriol with or without stimulation of cells with IL-1ß or TNF-α. The proliferation of cells was studied by MTT assay. Levels of cytokines were analyzed by a magnetic bead-based multiplex assay (a panel of 27 important cytokines and IL-6 alone) and RT-PCR was used to validate the concentrations of the key cytokines secreted by SSC. The vitamin D receptor (VDR) was visualized by immunofluorescence in SSC and by immunohistochemistry in the synovial tissues of three RA and three OA patients. RESULTS: We detected intense staining for VDR in the synovial lining and vascular endothelium in tissue sections from all our RA and OA patients. Both 1,25(OH)2D3 and calcipotriol inhibited SSC proliferation for a prolonged time (up to 23 days with calcipotriol), but dexamethasone tended to increase SSC proliferation in a 4-day culture. 1,25(OH)2D3, calcipotriol and dexamethasone reduced the secretion of most inflammatory factors. Calcipotriol and dexamethasone additively reduced the secretions of IL-6, IFN-γ, basic FGF and VEGF in TNF-α stimulated SSC. The level of IL-6 was still diminished at 10 days after exposure, emphasizing the long-term impact of calcipotriol on SSC. CONCLUSIONS: Exposure for 24-48h to 1,25(OH)2D3 or calcipotriol causes a long-lasting inhibition of cell proliferation and cytokine production in SSC in vitro.

Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue.

BACKGROUND: Seropositive rheumatoid arthritis (RA) is characterized by autoantibodies binding to citrullinated and homocitrullinated proteins. We wanted to study the expression patterns of these disease-associated protein forms and if the rheumatoid nodule and synovial tissue itself contain biologically active levels of citrullinating peptidyl arginine deiminases 2, 3 and 4 and homocitrullination-facilitating neutrophil enzyme myeloperoxidase. METHOD: Total of 195 synovial samples from metatarsal joints from five ACPA/RF-positive RA patients (n = 77), synovial samples from knees of eight seropositive RA (n = 60), seven seronegative RA (n = 33) and five osteoarthritis (n = 25) patients were analyzed for citrulline and homocitrulline contents using HPLC. The location of citrulline- and homocitrulline-containing proteins, PAD 2, 3, 4 and myeloperoxidase were shown by immunostaining. Myeloperoxidase and citrulline- or homocitrulline-containing proteins were stained on Western blot. RESULTS: Overall, necrosis was frequent in metatarsals of seropositive RA and absent in seronegative RA and osteoarthritis patients. In histological analysis, there was a significant local patterning and variation in the citrulline and homocitrulline content and it was highest in metatarsal synovial tissues of seropositive RA patients. We found peptidyl arginine deiminase 2, 3 and 4 in the lining and sublining layers of intact synovial tissue. Myeloperoxidase was found locally around necrotic areas. The tissues with necrosis contained the highest levels of citrulline and homocitrulline. CONCLUSIONS: Rheumatoid nodules and synovia contain significant amount of PAD2, 3 and 4 and myeloperoxidase enzymes. These enzymes could explain the levels of citrulline and homocitrulline in seropositive RA synovial and rheumatoid nodule tissues especially around necrotic tissue.

Presence of a truncated form of the vitamin D receptor (VDR) in a strain of VDR-knockout mice.

As part of our studies on the membrane-initiated actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [(3)H]-1alpha,25(OH)(2)D(3) in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5-50% of that seen in wild-type animals) specific binding of [(3)H]-1alpha,25(OH)(2)D(3) in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3-9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [(3)H]-1alpha,25(OH)(2)D(3) binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.

The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin D3 in vivo and in vitro.

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3.

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.