Search

Glioma is often referred to as one of the most dreadful central nervous system (CNS)-specific tumors with rapidly-proliferating cancerous glial cells, accounting for nearly half of the brain tumors at an annual incidence rate of 30-80 per a million population. Although glioma treatment remains a significant challenge for researchers and clinicians, the rapid development of nanomedicine provides tremendous opportunities for long-term glioma therapy. However, several obstacles impede the development of novel therapeutics, such as the very tight blood-brain barrier (BBB), undesirable hypoxia, and complex tumor microenvironment (TME). Several efforts have been dedicated to exploring various nanoformulations for improving BBB permeation and precise tumor ablation to address these challenges. Initially, this article briefly introduces glioma classification and various pathogenic factors. Further, currently available therapeutic approaches are illustrated in detail, including traditional chemotherapy, radiotherapy, and surgical practices. Then, different innovative treatment strategies, such as tumor-treating fields, gene therapy, immunotherapy, and phototherapy, are emphasized. In conclusion, we summarize the article with interesting perspectives, providing suggestions for future glioma diagnosis and therapy improvement.

Photodynamic therapy (PDT) is a non-invasive or minimally-invasive treatment which applies photosensitizers (PSs) to create reactive oxygen species (ROS) exposed to light trigger to destroy cancer cells. PDT can activate host anti-tumor immune responses but not powerful enough to kill metastatic tumors. Because of its carrier advantage, imaging, and therapeutic function together with enhanced permeability and retention (EPR) effect, nano-materials have already been used in photo-immunotherapy. Herein, photodynamic immunotherapy (PDIT) based on nanotechnology seems to be a hopeful new form of cancer therapy. In this article, we firstly summarize the recent development in photodynamic immunotherapy based on nanotechnology.

Photodynamic therapy (PDT) has been extensively investigated for decades for tumor treatment because of its non-invasiveness, spatiotemporal selectivity, lower side-effects, and immune activation ability. It can be a promising treatment modality in several medical fields, including oncology, immunology, urology, dermatology, ophthalmology, cardiology, pneumology, and dentistry. Nevertheless, the clinical application of PDT is largely restricted by the drawbacks of traditional photosensitizers, limited tissue penetrability of light, inefficient induction of tumor cell death, tumor resistance to the therapy, and the severe pain induced by the therapy. Recently, various photosensitizer formulations and therapy strategies have been developed to overcome these barriers. Significantly, the introduction of nanomaterials in PDT, as carriers or photosensitizers, may overcome the drawbacks of traditional photosensitizers. Based on this, nanocomposites excited by various light sources are applied in the PDT of deep-seated tumors. Modulation of cell death pathways with co-delivered reagents promotes PDT induced tumor cell death. Relief of tumor resistance to PDT with combined therapy strategies further promotes tumor inhibition. Also, the optimization of photosensitizer formulations and therapy procedures reduces pain in PDT. Here, a systematic summary of recent advances in the fabrication of photosensitizers and the design of therapy strategies to overcome barriers in PDT is presented. Several aspects important for the clinical application of PDT in cancer treatment are also discussed.

Differentiated thyroid carcinoma (DTC) is the most common endocrine malignancy and highly expresses the receptor for 1,25-dihydroxyvitamin D (1,25(OH)2D). However, it is unclear whether 1,25(OH)2D regulates DTC proliferation and differentiation. Here, we found that 1,25(OH)2D3 inhibited proliferation but not differentiation of the DTC cells. Notably, CYP27B1was elevated in DTC cells and 25-hydroxyvitamin D3 (25(OH)D3) reduced DTC cell proliferation. Knockdown of VDR did not affect the anti-proliferative effects of 1,25(OH)2D3. However, knockdown of CCAAT enhancer-binding protein β (C/EBPβ)abolished 1,25(OH)2D3-suppressed DTC cell proliferation. In addition, 1,25(OH)2D3 induced phosphorylation and translocation of C/EBPβto the nucleus from the cytoplasm. However, inhibition of p38 mitogen-activated protein kinases (MAPK) abrogated 1,25(OH)2D3-induced phosphorylation and nuclear translocation of C/EBPβas well as 1,25(OH)2D3-suppressed DTC cell proliferation. Knockdown of C/EBPβreduced the expression of Notch3. Knockdown of Notch3 blocked 1,25(OH)2D3-suppressed DTC cell proliferation. In the DTC cell-derived xenograft SCID mouse, knockdown of C/EBPβmarkedly increased tumor growth and proliferation and decreased apoptosis. In DTC patients, C/EBPβwas predominantly located in the cytoplasm of DTC cells in the tumor tissue when compared with adjacent non-cancerous tissue in which C/EBPβis located in the nucleus. In conclusion, C/EBPβstimulated Notch3signaling via the p38 MAPK-dependent pathway mediates the inhibitory effect of 1,25(OH)2D on DTC cell proliferation.

In recent years, nanomaterials and nanotechnology have emerged as vital factors in the medical field with a unique contribution to cancer medicine. Given the increasing number of cancer patients, it is necessarily required to develop innovative strategies and therapeutic modalities to tackle hypoxia, which forms a hallmark and great barrier in treating solid tumors. The present review details the challenges in nanotechnology-based hypoxia, targeting the strategies and solutions for better therapeutic performances. The interaction between hypoxia and tumor is firstly introduced. Then, we review the recently developed engineered nanomaterials towards multimodal hypoxia tumor therapies, including chemotherapy, radiotherapy, and sonodynamic treatment. In the next part, we summarize the nanotechnology-based strategies for overcoming hypoxia problems. Finally, current challenges and future directions are proposed for successfully overcoming the hypoxia tumor problems.

Wonderful black phosphorus (BP) and some BP analogs (BPAs) have been increasingly studied for their biomedical applications owing to their fascinating properties and biodegradability, but opportunities and challenges have always coexisted in their study. Poor stability upon exposure to the natural environment is the major obstacle hampering their in vivo applications. BP/polymer and BPAs/polymer nanocomposites can not only efficiently prevent their oxidation and aggregation but also exhibit "biological activity" due to synergistic effects. In this review, we briefly describe the synthesis methods and stability strategies of BP/polymer and BPAs/polymer. Then, advances pertaining to their exciting therapeutic applications in various fields are systematically introduced, such as cancer therapy (phototherapy, drug delivery, and synergistic immunotherapy), bone regeneration, and neurogenesis. Some challenges for future clinical trials and possible directions for further study are finally discussed.

Thyroid cancer has the fastest rising incidence among cancers, especially for differentiated thyroid carcinoma (DTC). Although the prognosis of DTC is relatively good, if it changes to anaplastic thyroid carcinoma (ATC), the prognosis will be very poor. The prognosis of DTC is largely depending on the degree of cell differentiation and proliferation. However, whether the vitamin D receptor (VDR) plays a role in regulating the proliferation and the differentiation of DTC cells is unclear. In the present study, we found that VDR was upregulated in DTC tissues compared to the adjacent non-cancerous tissue. Knockdown of VDR increased proliferation and decreased differentiation proliferation in DTC cells in vitro as well as DTC cell-derived xenografts in vivo. In contrast, overexpression of VDR had an opposite effect. Knockdown of E-cadherin abolished VDR-induced suppression of proliferation and enhancement of differentiation of the DTC cells. Knockdown of β-catenin partially reversed the effect of the VDR knockdown. VDR increases the levels of E-cadherin in the plasma membrane and decreases the levels of β-catenin in the nucleus. VDR binds to E-cadherin and β-catenin in the plasma membrane of the DTC cell. Taken together, VDR inhibits DTC cell proliferation and promotes differentiation via regulation of the E-cadherin/β-catenin complex, potentially representing novel clues for a therapeutic strategy to attenuate thyroid cancer progression.

Summary In this population-based, cross-sectional study, we investigated vertebral fracture (VF) prevalence among Chinese postmenopausal women. We found 14.7% of population had VFs, which increased with age. Age ≥ 65 years, hip fracture, and densitometric osteoporosis were significantly associated with VFs. The prevalence of osteoporosis was remarkably high. Purpose To investigate VF prevalence among Chinese postmenopausal women in this population-based, randomized-sampling, cross-sectional study. Methods The investigator obtained lists of women from communities. Randomization was performed using SAS programming based on age group in each region. Postmenopausal women aged ≥ 50 years in the urban community were included. The investigator interviewed subjects to collect self-reported data and measured BMD. Spine radiographs were adjudicated by Genant’s semi-quantitative method. VFs were defined as fractures of at least one vertebra classified by Genant’s score 1–3 and were analyzed using descriptive statistics. Results A total of 31,205 women listed for randomized sampling from 10 Tier-3 hospitals at 5 regions. Of 2634 women in the full analysis set, 14.7% (388/2634, 95% CI: 13.4, 17.1) had prevalent VFs. VF prevalence increased with age (Cochran–Armitage test p Conclusion VFs are prevalent among Chinese postmenopausal women who were ≥ 50 years and community-dwelled. Osteoporosis prevalence is remarkable when fragile fractures were part of clinical diagnosis.

Optoelectronic science and 2D nanomaterial technologies are currently at the forefront of multidisciplinary research and have numerous applications in electronics and photonics. The unique energy and optically induced interfacial electron transfer in these nanomaterials, enabled by their relative band alignment characteristics, can provide important therapeutic modalities for healthcare. Given that nano-heterostructures can facilitate photoinduced electron-hole separation and enhance generation of reactive oxygen species (ROS), 2D nano-heterostructure-based photosensitizers can provide a major advancement in photodynamic therapy (PDT), to overcome the current limitations in hypoxic tumor microenvironments. Herein, a bismuthene/bismuth oxide (Bi/BiOx)-based lateral nano-heterostructure synthesized using a regioselective oxidation process is introduced, which, upon irradiation at 660 nm, effectively generates 1 O2 under normoxia but produces cytotoxic •OH and H2 under hypoxia, which synergistically enhances PDT. Furthermore, this Bi/BiOx nano-heterostructure is biocompatible and biodegradable, and, with the surface molecular engineering used here, it improves tumor tissue penetration and increases cellular uptake during in vitro and in vivo experiments, yielding excellent oxygen-independent tumor ablation with 660 nm irradiation, when compared with traditional PDT agents.

Background Fragility fracture is associated with bone mineral density (BMD), and most databases used in related researches are instrument-matched. Little is known about the relationship between BMD and fragility fracture risk of native Chinese, especially using local databases as reference databases. Objective To investigate relationship between BMD and risk of fragility fracture in native China. Methods 3,324 cases, including 2,423 women (67.7 ± 8.9 years) and 901 men (68.4 ± 11.6 years) having radiological fragility fractures and 3,324 age- and gender-matched controls participated in the study. We measured BMD at posteroanterior spine and hip using dual-energy X-ray absorptiometry (DXA), calculated BMD measurement parameters based on our own BMD reference database. Results BMDs and mean T-scores were lower in case group (with clinical fragility) than in control group (without clinical fragility). In patients with fragility fractures, prevalence of lumbar osteoporosis, low bone mass, and normal BMD were 78.9 %, 19.3 %, and 1.8 %, respectively, in women, and 49.5, 44.8 %, and 5.7 %, respectively, in men. In hip, these prevalence rates were 67.2 %, 28.4 %, and 4.4 % in females, and 43.2 %, 45.9 %, and 10.9 % in males, respectively, showing differences between females and males. Multivariate Cox regression analysis showed that after adjusting age, height, weight, and body mass index, fracture hazard ratio (HR) increased by 2.7–2.8 times (95 % CI 2.5–3.1) and 3.6–4.1 times (95 %CI 3.0–5.1) for women and men respectively with decreasing BMD parameters. In both sexes, risk of fragility fracture increased approximately 1.6–1.7 times (95 % CI 1.5–1.8) for every 1 T-score reduction in BMD. Conclusions Risk of clinical fragility fracture increases with decreasing BMD measurement parameters and anthropometric indicators in native China, and fracture HR varies from gender and site.

Photothermal therapy (PTT) based on two-dimensional (2D) nanomaterials has shown significant potential in cancer treatment. However, developing 2D nanomaterial-based theranostic agents with good biocompatibility and high therapeutic efficiency remains a key challenge. Bulk titanium (Ti) has been widely used as biomedical materials for their reputable biocompatibility, whereas nanosized Ti with a biological function remains unexplored. In this work, the 2D Ti nanosheets (NSs) are successfully exfoliated from nonlayer bulk Ti and utilized as an efficient theranostic nanoplatform for dual-modal computed tomography/photoacoustic (CT/PA) imaging-navigated PTT. Besides the excellent biocompatibility obtained by TiNSs as expected, they are found to show strong absorption ability with an extinction coefficient of 20.8 L g-1 cm-1 and high photothermal conversion ability with an efficiency of 61.5% owing to localized surface plasmon resonances, which exceeds most of other well-known photothermal agents, making it quite promising for PTT against cancer. Furthermore, the metallic property and light-heat-acoustic transformation endow 2D Ti with the strong CT/PA imaging signal and efficient cancer therapy, simultaneously. This work highlights the enormous potential of nanosized Ti in both the diagnosis and treatment of cancer. As a paradigm, this study also paves a new avenue for the elemental transition-metal-based cancer theranostics.

Basal-like breast cancer exhibits a triple-negative phenotype and has a poor prognosis, even with traditional chemical and anti-human epidermal growth factor receptor (HER) treatments. However, the high mutation rate of this obstinate cancer type renders it suitable for immunotherapy. Photothermal therapy (PTT) is a high-efficiency method for inducing tumor neoantigen release in situ, which has great potential for use in cancer immunotherapy. Here, we prepared a biomimetic black phosphorus quantum dot (BPQDs) formulation to induce breast cancer cell apoptosis in situ by near-infrared (NIR) laser irradiation to mobilize the immune system to eliminate the residual and metastatic cancer cells. Erythrocyte membranes (RMs) were used to coat the BPQDs, forming a BPQD-RM nanovesicle (BPQD-RMNV) biomimetic formulation that exhibited a long circulation time and tumor accumulation in vivo. The basal-like 4T1 breast tumor underwent apoptosis and necrosis with the irradiation and recruited dendritic cells (DCs) to capture the tumor antigens in vivo. Furthermore, programmed cell death protein 1 (PD-1) antibody (aPD-1) was employed to prevent the CD8+ T cells from exhaustion. Notably, BPQD-RMNV-mediated PTT combined with aPD-1 treatment significantly delayed residual and metastatic tumor growth in vivo. Hence, BPQD-RMNV-mediated PTT combined with immune checkpoint blockade antibody increased the infiltration and activity of CD8+ T cells in the tumor, which directly restrained basal-like breast tumor growth in vivo.

Summary Introduction Vitamin D supplementation has been widely recommended to prevent falls. However, considerable controversy exists regarding the association of such supplementation and fall risk. Previous meta-analyses yielded inconsistent results because of differences in the baseline of 25-hydroxyvitamin D [25(OH)D] and dose of vitamin D and use of vitamin D or in combination with calcium in different studies. Furthermore, some studies published recently were not included in the previous meta-analyses. Therefore, an updated and comprehensive meta-analysis is warranted. Methods We systematically searched several literature databases including PubMed and the Embase from inception to September 2020. The protocol for this meta-analysis was registered with PROSPERO (CRD42021226380). Randomized clinical trials (RCTs) reporting the effect of vitamin D supplementation alone or with calcium on fall incidence were selected from studies. Qualitative and quantitative information was extracted; the random-effects model was conducted to pool the data for fall; statistical heterogeneity was assessed using the I2 test and potential for publication bias was assessed qualitatively by a visual estimate of the funnel plot and quantitatively by calculation of the Begg's test and the Egger's test. Results Of the citations retrieved, 31 eligible studies involving 57 867 participants met inclusion criteria, reporting 17 623 falls. A total of 21 RCTs of vitamin D alone and 10 RCTs of vitamin D plus calcium were included in the meta-analysis. The meta-analysis of 21 RCTs (51 984 participants) of vitamin D supplementation alone (daily or intermittent doses of 400–60 000 IU) did not show a reduced risk of falls (The risk ratio [RR] 1.00, 95% confidence intervals [CI] 0.95 to 1.05) compared to placebo or no treatment. Subgroup analyses showed that the baseline of serum 25(OH)D concentration less than 50 nmol/L resulted in a reduction of fall risk (RR 0.77, 95% CI 0.61 to 0.98). In contrast, the meta-analysis of 10 RCTs (5883 participants) of combined supplementation of vitamin D (daily doses of 700–1000 IU) and calcium (daily doses of 1000–1200 mg) showed a 12% reduction in the risk of fall (RR 0.88, 95% CI 0.80 to 0.97). Conclusions The combination of vitamin D and calcium have beneficial effects on prevention falls in old adults. Although vitamin D supplementation alone has no effect on fall risk in old adults with 25(OH)D levels higher than 50 nmol/L, vitamin D supplementation alone does have a benefit on prevention of falls in old adults with 25(OH)D levels lower than 50 nmol/L.

The aim was to compare the National Osteoporosis Foundation (NOF) and the UK National Osteoporosis Guideline Group (NOGG) guidelines for the detection of vertebral fractures in postmenopausal Chinese women. The NOF guidelines had higher accuracy than the UK guidelines for spinal radiography examination. To compare the National Osteoporosis Foundation (NOF) and the UK National Osteoporosis Guideline Group (NOGG) guidelines for the detection of vertebral fractures in postmenopausal Chinese women. A cross-sectional study on 255 community-dwelling postmenopausal women was conducted in Hunan province in 2017. Demographic and clinical characteristics and risk factors were recorded through questionnaires. Height and weight were measured using standard methods, and bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry. Vertebral fractures were diagnosed by radiography using semi-quantitative morphometry. The prevalence of vertebral fractures was 9.4%. Women with vertebral fractures were significantly older and shorter, had higher years-since menopause and height loss values and lower BMD and T-scores at the femoral neck and total hip, and were more likely to have a history of previous fractures. The sensitivity and specificity of the NOF guidelines were 91.7% and 39.8%, respectively, while the NOGG guidelines had lower sensitivity (87.5%) and specificity (32.5%). However, a higher percentage had indications for radiography in the 50-64 years age group according to the NOGG guidelines, while a higher percentage had indications for radiography in the ≥ 65 year group according to the NOF guidelines. The NOF guidelines were better than the NOGG guidelines for spinal radiography examination in the Chinese postmenopausal women.

The present study demonstrated that the lower-extremity muscle performance in Chinese community-dwelling postmenopausal women with lower bone mineral density (BMD) was positively associated with hip BMD and negatively associated with estimated fracture risk. Postmenopausal women are at high risk for osteoporotic fractures. It has been shown that decreased lower-extremity muscle performance is associated with osteoporotic fractures. However, the relationship between muscle performance and bone mineral density in postmenopausal women is inconsistent in the literature. The present study was to investigate the relationship between lower-extremity muscle performance and BMD or estimated fracture risk in community-dwelling postmenopausal women. Two hundred forty-seven postmenopausal women aged 50–85 years were recruited in the study. The short physical performance battery (SPPB) tool including the chair stand test (CST), gait speed test (GST), and balance test (BT) was used to determine lower-extremity functioning and the CST, GST, BT, and SPPB total scores were recorded. The BMD of lumbar spine (LSBMD), femoral neck (FNBMD), and total hip (THBMD) were measured by dual-energy X-ray absorptiometry (DXA), and the vertebral fracture was confirmed by lateral spine X-rays radiographs. In addition, patients’ 10-year estimated major osteoporotic fracture risk (MOFR) and hip fracture risk (HFR) were assessed by the Fracture Risk Assessment Tool (FRAX). Linear regression analysis was used to analyze the association between muscle performance and BMD. The CST, GST, and SPPB total scores were positively associated with LSBMD, THBMD, and FNBMD before adjustment for age, height, and weight. The SPPB total score was positively associated with FNBMD and THBMD, but not with LSBMD after adjustment for age, height, and weight. The BT score was positively associated with FNBMD and THBMD, but not with LSBMD before and after adjustment for age, height, and weight. Moreover, the CST, GST, BT, and SPPB scores were negatively associated with the FRAX score. The lower-extremity muscle performance in community-dwelling postmenopausal women is positively associated with FNBMD and THBMD and negatively associated with the FRAX score.

The clustered regularly interspaced short palindromic repeats (CRISPR) molecular system has emerged as a promising technology for the detection of nucleic acids. Herein, the development of a surface plasmon resonance (SPR) sensor that is functionalized with a layer of locally grown graphdiyne film, achieving excellent sensing performance when coupled with catalytically deactivated CRISPR-associated protein 9 (dCas9), is reported. dCas9 protein is immobilized on the sensor surface and complexed with a specific single-guide RNA, enabling the amplification-free detection of target sequences within genomic DNA. The sensor, termed CRISPR-SPR-Chip, is used to successfully analyze recombinant plasmids with only three-base mutations with a limit of detection as low as 1.3 fM. Real-time monitoring CRISPR-SPR-Chip is used to analyze clinical samples of patients with Duchenne muscular dystrophy with two exon deletions, which are detected without any pre-amplification step, yielding significantly positive results within 5 min. The ability of this novel CRISPR-empowered SPR (CRISPR-eSPR) sensing platform to rapidly, precisely, sensitively, and specifically detect a target gene sequence provides a new on-chip optic approach for clinical gene analysis.

The diagnosis and management of osteoporosis and osteoporotic fractures are challenging in rural and underdeveloped areas of China because medical resources are inaccessible; thus, a simple and accurate method is essential for the detection of vertebral fractures. We aimed to examine the relationship between historical height loss (HHL) and vertebral fractures in postmenopausal Chinese women.A cross-sectional study of 255 postmenopausal women aged 50 years or older was conducted in September 2017. Demographic data, including self-reported tallest historical height and current height were analyzed. Vertebral fractures were assessed using X-ray radiography and HHL thresholds were examined using specificity and sensitivity testing.The average age of the 255 participants was 66.3 ± 9.0 years and their mean HHL was 3.5 ± 2.8 cm. The 24 women who were found to have vertebral fractures were older, had more years since menopause (YSM), and a larger HHL compared to those without vertebral fractures. Logistic regression analysis showed that age was a better predictor of vertebral fractures than HHL was, and the cutoff age for detecting vertebral fractures was 71 years, with an area under the receiver operating characteristic curve of 0.750.Although the women in this study with vertebral fractures had a greater height loss than those without fractures, it was apparent that age, rather than HHL, is the best way to determine who is most likely to develop vertebral fractures.

Optical techniques using developed laser and optical devices have made a profound impact on modern medicine, with "biomedical optics" becoming an emerging field. Sophisticated technologies have been developed in cancer nanomedicine, such as photothermal therapy and photodynamic therapy, among others. However, single-mode phototherapy cannot completely treat persistent tumors, with the challenges of relapse or metastasis remaining; therefore, combinatorial strategies are being developed. In this review, the role of light in cancer therapy and the challenges of phototherapy are discussed. The development of combinatorial strategies with other therapeutic methods, including chemotherapy, immunotherapy, gene therapy, and radiotherapy, is presented and future directions are further discussed. This review aims to highlight the significance of light in cancer therapy and discuss the combinatorial strategies that show promise in addressing the challenges of phototherapy.

We sought to determine the long-term bioavailability of single doses of intramuscular (IM) vita-min DForty healthy volunteers with hypovitaminosis D received a single dose of 200,000, 400,000, or 600,000 IU intramuscular DAfter a single dose of DA single dose of 200,000 IU, 400,000 IU, or 600,000 IU IM D

This review focuses on the biologic importance of the vitamin D binding protein (DBP) with emphasis on its regulation of total and free vitamin D metabolite levels in various clinical conditions. Nearly all DBP is produced in the liver, where its regulation is influenced by estrogen, glucocorticoids and inflammatory cytokines but not by vitamin D itself. DBP is the most polymorphic protein known, and different DBP alleles can have substantial impact on its biologic functions. The three most common alleles-Gc1f, Gc1s, Gc2-differ in their affinity with the vitamin D metabolites and have been variably associated with a number of clinical conditions. Although DBP has a number of biologic functions independent of vitamin D, its major biologic function is that of regulating circulating free and total levels of vitamin D metabolites. 25 hydroxyvitamin D (25(OH)D) is the best studied form of vitamin D as it provides the best measure of vitamin D status. In a normal non-pregnant individual, approximately 0.03% of 25(OH)D is free; 85% is bound to DBP, 15% is bound to albumin. The free hormone hypothesis postulates that only free 25(OH)D can enter cells. This hypothesis is supported by the observation that mice lacking DBP, and therefore with essentially undetectable 25(OH)D levels, do not show signs of vitamin D deficiency unless put on a vitamin D deficient diet. Similar observations have recently been described in a family with a DBP mutation. This hypothesis also applies to other protein bound lipophilic hormones including glucocorticoids, sex steroids, and thyroid hormone. However, tissues expressing the megalin/cubilin complex, such as the kidney, have the capability of taking up 25(OH)D still bound to DBP, but most tissues rely on the free level. Attempts to calculate the free level using affinity constants generated in a normal individual along with measurement of DBP and total 25(OH)D have not accurately reflected directly measured free levels in a number of clinical conditions. In this review, we examine the impact of different clinical conditions as well as different DBP alleles on the relationship between total and free 25(OH)D, using only data in which the free 25(OH)D level was directly measured. The major conclusion is that a number of clinical conditions alter this relationship, raising the question whether measuring just total 25(OH)D might be misleading regarding the assessment of vitamin D status, and such assessment might be improved by measuring free 25(OH)D instead of or in addition to total 25(OH)D.

Renal 25-hydroxyvitamin D-1α-hydroxylase (1αOHase, CYP27B1) and 24-hydroxylase (24OHase, CYP24A1) are tightly regulated. However, little is known about the regulation of 1α(OH)ase and 24(OH)ase in extrarenal tissue such as the epidermis. This study was to determine the roles of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF 23) in the regulation of 1α(OH)ase and 24(OH)ase in epidermal keratinocytes as well as epidermal keratinocyte proliferation and differentiation. The results showed that PTH increased the protein level of 1α(OH)ase in human epidermal keratinocyte cell line HaCaT, but had no effect on the level of 24(OH)ase. The effect of PTH on 1α(OH)ase was blocked by the PKC inhibitor. Treatment with FGF23 decreased mRNA and protein levels of 1α(OH)ase and increased mRNA and protein levels of 24(OH)ase in HaCaT cells. The effect of FGF23 on 1α(OH)ase and 24(OH)ase was blocked by the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) inhibitor. In addition, treatment with PTH enhanced levels of differentiation markers including keratin 1, involucrin, loricrin, and filaggrin but reduced levels of BrdU incorporation in HaCaT cells. These effects were inhibited by the PKC inhibitor. FGF23 enhanced proliferation of HaCaT cells, but reduced levels of early differentiation markers including keratin 1 and involucrin and enhanced levels of the later differentiation markers including loricrin and filaggrin. These results suggest that PTH stimulates 1α(OH)ase expression and differentiation of HaCaT cells and inhibits proliferation via PKC. The data also suggest that FGF23 inhibits 1α(OH)ase expression and stimulates 24(OH)ase expression via MAPK/ERK. In addition, FGF23 enhances proliferation and late differentiation and inhibits early differentiation of HaCaT keratinocytes.

Due to their prominent physicochemical properties, 2D materials are broadly applied in biomedicine. Currently, 2D materials have achieved great success in treating many diseases such as cancer and tissue engineering as well as bone therapy. Based on their different characteristics, 2D materials could function in various ways in different bone diseases. Herein, the application of 2D materials in bone tissue engineering, joint lubrication, infection of orthopedic implants, bone tumors, and osteoarthritis are firstly reviewed comprehensively together. Meanwhile, different mechanisms by which 2D materials function in each disease reviewed below are also reviewed in detail, which in turn reveals the versatile functions and application of 2D materials. At last, the outlook on how to further broaden applications of 2D materials in bone therapies based on their excellent properties is also discussed.

Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1αis recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1αand the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1αactivation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1αand induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1αactivation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1αcomplex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation.

Photodynamic therapy (PDT), as a non-invasive therapeutic modality that is alternative to radiotherapy and chemotherapy, is extensively investigated for cancer treatments. Although conventional organic photosensitizers (PSs) are still widely used and have achieved great progresses in PDT, the disadvantages such as hydrophobicity, poor stability within PDT environment and low cell/tissue specificity largely limit their clinical applications. Consequently, nano-agents with promising physicochemical and optical properties have emerged as an attractive alternative to overcome these drawbacks of traditional PSs. Herein, the up-to-date advances in the fabrication and fascinating applications of various nanomaterials in PDT have been summarized, including various types of nanoparticles, carbon-based nanomaterials, and two-dimensional nanomaterials, etc. In addition, the current challenges for the clinical use of PDT, and the corresponding strategies to address these issues, as well as future perspectives on further improvement of PDT have also been discussed.

p120-catenin (p120) is an important regulator in the function and stability of E-cadherin. However, the role of p120 in the epidermis is unclear. Previous studies have shown that globally knockout of p120 caused increased epidermal proliferation but little changes in epidermal differentiation and permeability. In the present study, we generated a conditional knockout mouse model and examined epidermal proliferation, differentiation and permeability. The results showed that conditional knockout of p120 in the epidermis caused not only increased epidermal proliferation but also decreased epidermal differentiation and increased permeability. These data suggest that p120 is required for suppressing epidermal proliferation, promoting epidermal differentiation and maintaining permeability barrier function of the epidermis.

Black phosphorus (BP) has recently emerged as an intriguing photothermal agent in photothermal therapy (PTT) against cancer by virtue of its high photothermal efficiency, biocompatibility, and biodegradability. However, naked BP is intrinsically characterized by easy oxidation (or natural degradation) and sedimentation inside the tumor microenvironment, leading to a short-term therapeutic and inhomogeneous photothermal effect. Development of BP-based nanocomposites for PTT against cancer therefore remains challenging. The present work demonstrates that green and injectable composite hydrogels based on cellulose and BP nanosheets (BPNSs) are of great efficiency for PTT against cancer. The resultant cellulose/BPNS-based hydrogel possesses 3D networks with irregular micrometer-sized pores and thin, strong cellulose-formed walls and exhibits an excellent photothermal response, enhanced stability, and good flexibility. Importantly, this hydrogel nanoplatform is totally harmless and biocompatible both in vivo and in vitro. This work may facilitate the development of BP-polymer-based photothermal agents in the form of hydrogels for biomedical-related clinic applications.

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-gamma1 (PLC-gamma1) is a downstream target of EGFR signaling, but the role and necessity of PLC-gamma1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-gamma1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-gamma1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-gamma1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-gamma1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-gamma1 is independent of its lipase activity.

Calcium induces epidermal keratinocyte differentiation, but the mechanism is not completely understood. We have previously demonstrated that calcium-induced human keratinocyte differentiation requires an intracellular calcium rise caused by phosphatidylinositol 3-kinase (PI3K)-dependent activation of phospholipase C-gamma1. In this study we sought to identify the upstream signaling pathway necessary for calcium activation of PI3K and its subsequent activation of phospholipase C-gamma1. We found that calcium induces the recruitment of PI3K to the E-cadherin-catenin complex at the plasma membrane of human keratinocytes. Knocking-down E-cadherin, beta-catenin, or p120-catenin expression blocked calcium activation of PI3K and phospholipase C-gamma1 and calcium-induced keratinocyte differentiation. However, knocking-down gamma-catenin expression had no effect. Calcium-induced PI3K recruitment to E-cadherin stabilized by p120-catenin at the plasma membrane requires beta-catenin but not gamma-catenin. These data indicate that the recruitment of PI3K to the E-cadherin/beta-catenin/p120-catenin complex via beta-catenin at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and, ultimately, keratinocyte differentiation.

Oral squamous cell carcinoma (OSCC) is one of the most lethal malignant tumors. The cadherin/catenin cell-cell adhesion complex plays a major role in cancer development and progression. p120-catenin (p120) is a cytoplasmic molecule closely associated with E-cadherin which activates phospholipase C-γ1 (PLC-γ1). Our previous studies indicate that activation of PLC-γ1 plays a critical role in epidermal growth factor (EGF)-induced migration and proliferation of squamous cell carcinoma (SCC) cells and phosphatidylinositol 3-kinase enhancer (PIKE) is highly expressed in SCC cells and mediates EGFR-dependent SCC cell proliferation. Our current study was to determine whether the expression of E-cadherin, p120, PLC-γ1, and PIKE, is associated with OSCC. To address this issue, we assessed levels and localization of E-cadherin, p120, PLC-γ1, and PIKE in specimen of 92 patients with OSCC by immunohistochemistry. The results showed that the expression of E-cadherin, and p120 negatively correlated with the tumor differentiation and the expression of PLC-γ1 and PIKE positively correlated with the tumor differentiation. The expression of PLC-γ1 and PIKE in OSCC stage T3 + T4 or in OSCC with lymph node metastasis was significantly higher than that in OSCC stage T1 + T2 or in OSCC without lymph node metastasis. The expression of p120 positively correlated with levels of E-cadherin but negatively correlated with levels of PLC-γ1 and PIKE in OSCC. These data indicate that increased expression of PLC-γ1 and PIKE and decreased expression of E-cadherin and p120 are associated with the aggressiveness of OSCC.

Calcium is a strong inducer of keratinocyte differentiation. We have previously demonstrated that extracellular calcium promotes keratinocyte differentiation via E-cadherin-catenin complex-mediated phospholipase C-γ1 (PLC-γ1) activation in the plasma membrane. However, it is unclear whether dietary calcium regulates keratinocyte proliferation, differentiation or carcinogenesis. To address this issue, the rates of oral tumor and levels of proliferation and differentiation in the oral epithelium were assessed in mice on different calcium diets and the carcinogen 4-nitroquinoline-1-oxide. The results showed that mice on the high calcium diet had lower rates of oral tumors, lower levels of proliferation and higher levels of differentiation in the normal oral epithelium than those on the normal calcium diet. Higher levels of E-cadherin, β-catenin, p120-catenin (p120), epidermal growth factor receptor (EGFR), and calcium and lower levels of PLC-γ1 were also noted in the normal oral epithelium in mice on high calcium diet than the control mice. In contrast, mice on low calcium diet had opposite effects. However, dietary calcium had no effect on the proliferation, differentiation or the levels of E-cadherin, β-catenin, p120, PLC-γ1 and EGFR in oral tumors. These data indicate that dietary calcium increases calcium levels in oral epithelium, suppresses oral carcinogenesis, inhibits proliferation and promotes differentiation of normal oral epithelium. Increased E-cadherin, β-catenin, p120 and EGFR and decreased PLC-γ1 may participate in the inhibitory effect of dietary calcium in oral carcinogenesis.

Vitamin D deficiency is reportedly linked to a variety of autoimmune diseases. However, the relationship between thyroid autoimmunity in Graves disease (GD) and vitamin D deficiency is unclear. The goal of this study was to determine whether increased thyroid hormone autoantibody titer is associated with vitamin D deficiency in GD patients.A total of 70 patients with GD and 70 matched control subjects were recruited to our study. The levels of 25-hydroxyvitamin D (25[OH]D), calcium, parathyroid hormone (PTH), free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), thyrotropin-receptor antibody (TRAb), thyroid-peroxidase antibody (TPOAb), and thyroglobulin antibody (TGAb) in serum collected from these patients and controls were examined.The level of 25(OH)D in serum from TRAb-positive GD patients was significantly lower than that in serum of healthy controls or TRAb-negative patients. However, compared with control subjects, the level of PTH in serum was increased in TRAb-positive GD patients. The rate of vitamin D deficiency (defined as serum 25[OH]D50 nmol/L) in TRAb-positive GD patients was significantly higher than in healthy controls or TRAb-negative GD patients. The level of 25(OH)D in serum was inversely correlated with TRAb titer in serum of TRAb-positive GD patients. However, our results did not show a correlation between 25(OH)D level and the levels of TPOAb, TGAb, FT3, FT4, or TSH.Low vitamin D status is associated with increased TRAb titer in GD, suggesting a possible link between vitamin D status and increased thyroid autoimmunity in GD patients.

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of skin. Its expression is stimulated by a number of agents including calcium and 1,25-dihydroxy-vitamin D3 that promote the differentiation process in keratinocytes. Within the distal regulatory region of the involucrin promoter lies an AP-1 site and an element homologous to other vitamin D response elements. In previous studies mutation of the AP-1 site was found to reduce basal activity and block calcium stimulation of the involucrin promoter, whereas the vitamin D response element was not critical for calcium regulation. In this study both elements proved to be important for 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. Mutation of the AP-1 site reduced basal activity and blocked 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. In contrast, mutation of the vitamin D response element did not reduce basal expression of the involucrin promoter or prevent calcium stimulation of involucrin gene expression, but blocked 1,25-dihydroxyvitamin D3 stimulation. The vitamin D response element from the involucrin gene bound the vitamin D receptor and the retinoid X receptor, but not the retinoic acid receptor, in a specific manner. We conclude that the AP-1 site and the vitamin D response element in the involucrin promoter play important roles in mediating the action of 1,25-dihydroxyvitamin D3 on involucrin expression, but the vitamin D response element provides specificity for the 1,25-dihydroxyvitamin D3 response lacking at the AP-1 site.

OBJECTIVES Recent studies suggest that serum lipid profiles are related to bone mineral density (BMD). But data about this relationship on Chinese population are scarce. We investigated the relationships between serum lipid and BMD in postmenopausal Chinese women. METHODS A cross-sectional study was conducted in 790 Chinese postmenopausal women. BMDs were measured by dual X-ray absorptiometry. Serum lipid profiles were obtained after a 12-h fasting. RESULTS Women with serum high-density lipoprotein cholesterol (HDL-C) levels of at least 1·55 mmol/l had a greater prevalence of osteoporosis compared with women with lower HDL-C (≤1·54 mmol/l). After controlling for age, menopausal duration, body mass index, serum creatinine levels, outdoor activity, smoking and alcohol intake, high HDL-C levels were associated with osteoporosis (OR = 1·64, 95%CI 1·16-2·33, P < 0·01). BMD at femoral neck and total hip was significantly lower in the higher HDL-C class than the lower class (0·722 ± 0·118 vs 0·744 ± 0·120 g/cm(2) , P < 0·01; 0·800 ± 0·126 vs 0·824 ± 0·125 g/cm(2) , P < 0·01, respectively). No association was found between total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) with BMD. CONCLUSIONS In Chinese postmenopausal women, elevated levels of serum HDL-C had a greater probability of being osteoporosis than the lower HDL-C levels. Our analysis showed higher HDL-C level that is favourable for cardiovascular diseases should be regarded as a risk factor for osteoporosis.

Keratinocytes produce vitamin D3 and convert it to the most active form, 1,25-dihydroxyvitamin D3, which regulates keratinocyte proliferation and differentiation. Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by 1,25-dihydroxyvitamin D3. Therefore, phospholipase C-gamma1 might be important in the signaling pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation. To test this hypothesis, phospholipase C-gamma1 expression in human keratinocytes was reduced by transfecting the cells with an antisense phospholipase C-gamma1 construct and then evaluating the response of the keratinocyte differentiation markers involucrin and transglutaminase to 1,25-dihydroxyvitamin D3. The results showed that involucrin and transglutaminase protein and mRNA levels were markedly reduced in keratinocytes transfected by the antisense phospholipase C-gamma1 construct. Cotransfection of keratinocytes with the involucrin or transglutaminase promoter construct and the antisense phospholipase C-gamma1 construct showed decreased involucrin or transglutaminase promoter activity in response to 1,25-dihydroxyvitamin D3. To further investigate the mechanism by which phospholipase C-gamma1 regulates keratinocyte differentiation, the calcium and inositol triphosphate levels in keratinocytes transfected by the antisense phospholipase C-gamma1 construct were measured following 1,25-dihydroxyvitamin D3 administration. The increase in keratinocyte intracellular free calcium and inositol triphosphate levels following 1,25-dihydroxyvitamin D3 administration were markedly reduced by the transfection of the antisense phospholipase C-gamma1 construct. These studies indicate that phospholipase C-gamma1 plays a critical role in the signal transduction pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation at least in part by mediating the increase in inositol triphosphate production and intracellular calcium mobilization following 1,25-dihydroxyvitamin D3 administration.

Calcium and 1,25 dihydroxyvitamin D (1,25(OH)(2)D) regulate the differentiation of keratinocytes. We have examined the mechanisms by which such regulation takes place, focusing primarily on the events leading to cornified envelope (CE) formation, in particular the mechanisms by which calcium and 1,25(OH)(2)D regulate the induction of involucrin, a component of the CE, and transglutaminase, the enzyme cross-linking involucrin and other substrates to form the CE. Both extracellular calcium (Ca(o)) and 1,25(OH)(2)D raise intracellular free calcium (Ca(i)) as a necessary step toward stimulating differentiation. Cells lacking the calcium sensing receptor (CaR) or phospholipase C-gamma 1 (PLC-gamma 1) fail to respond to Ca(o) or 1,25(OH)(2)D with respect to differentiation. Residing in the promoter of involucrin is a region responsive to calcium and 1,25(OH)(2)D, the calcium response element (CaRE). The CaRE contains an AP-1 site, mutations of which result in loss of responsiveness to Ca(o) and 1,25(OH)(2)D, indicating a role for protein kinases C (PKC). PKC alpha is the major PKC isozyme involved at least for calcium-induced differentiation. Thus, the regulation of keratinocyte differentiation by calcium and 1,25(OH)(2)D involves a number of signaling pathways including PLC and PKC activation, leading to the induction of proteins required for the differentiation process.

Phospholipase C-gamma1 (PLC-gamma1) is the most abundant member of the phospholipase C family expressed in human keratinocytes. PLC-gamma1 is induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) in normal keratinocytes via a DR6-type vitamin D responsive element. This regulation is not observed in transformed keratinocytes. The role of PLC-gamma1 in mediating 1alpha,25(OH)(2)D(3) and calcium-regulated differentiation was then tested. Both specific PLC inhibitors and antisense constructs which selectively block PLC-gamma1 production prevented 1alpha,25(OH)(2)D(3) and calcium from inducing markers of differentiation such as involucrin and transglutaminase. These studies demonstrate that PLC-gamma1 induction by 1alpha,25(OH)(2)D(3) is critical to the ability of this hormone to regulate keratinocyte differentiation.

Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by calcium. Phospholipase C-gamma1, therefore, may be involved in the signal transduction system leading to calcium regulation of keratinocyte differentiation. To test this hypothesis, expression of phospholipase C-gamma1 in human keratinocytes was blocked by transfecting cells with the antisense human phospholipase C-gamma1 cDNA construct. These cells demonstrated a dramatic reduction in phospholipase C-gamma1 protein level compared with the empty vector-transfected cells and a marked reduction in the mRNA and protein levels of the differentiation markers involucrin and transglutaminase following administration of calcium. Similarly, cotransfection of antisense phospholipase C-gamma1 constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters led to a substantial reduction in calcium-stimulated involucrin and transglutaminase promoter activities. Similar results were seen following treatment with a specific phospholipase C inhibitor U73122. To determine whether phospholipase C-gamma1 regulated differentiation by controlling intracellular calcium, we examined the ability of antisense phospholipase C-gamma1 to block the calcium-induced rise in intracellular calcium and found that it could. These findings indicate that phospholipase C-gamma1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium.

The 5'-flanking region of the human phospholipase C-gamma1 gene was isolated from a human P1 genomic DNA library. The S1-nuclease mapping and primer extension analysis revealed that there is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-gamma1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The fragment +135 to -877 in the 5'-flanking region of the human phospholipase C-gamma1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity and responded to 1,25-(OH)2D3 in transiently transfected human keratinocytes. Deletion and mutation studies of the fragment +135 to -877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases (DR6), AGGTCAgaccacTGGACA, located between -786 and -803 base pairs. Incubation of the oligonucleotide containing the DR6 with keratinocyte nuclear extracts produced a specific protein-DNA complex that shifted to a higher molecular weight form upon the addition of an antibody specific to the 1,25-(OH)2D3 receptor. Therefore, the 5'-flanking region of the human phospholipase C-gamma1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by 1, 25-(OH)2D3.

Phospholipase C-gamma1 (PLC-gamma1) is a multiple-domain protein and plays an important role in epidermal growth factor (EGF)-induced cell mitogenesis, but the underlying mechanism is unclear. We have previously demonstrated that PLC-gamma1 is required for EGF-induced mitogenesis of squamous cell carcinoma (SCC) cells, but the mitogenic function of PLC-gamma1 is independent of its lipase activity. Earlier studies suggest that the Src homology 3 (SH3) domain of PLC-gamma1 possesses mitogenic activity. In the present study, we sought to determine the role of the SH3 domain of PLC-gamma1 in EGF-induced SCC cell mitogenesis. We examined the effect of overexpression of PLC-gamma1, a catalytically active PLC-gamma1 mutant lacking the SH3 domain or a catalytically inactive PLC-gamma1 mutant lacking the X domain on EGF-induced SCC4 (tongue squamous cell carcinoma) cell mitogenesis. We found that overexpression of PLC-gamma1 enhanced EGF-induced SCC4 cell mitogenesis. This enhancement was abolished by deletion of the SH3 domain but not by deletion of the X catalytic domain. These data suggest that the SH3 domain, but not the catalytic domain, is required for PLC-gamma1 to mediate EGF-induced SCC4 cell mitogenesis.

Phospholipase C-gamma1 (PLC-gamma1), a tyrosine kinase substrate, has been implicated in the pathway for the epidermal growth factor receptor (EGFR)-induced cell migration. However, the underlying mechanism by which PLC-gamma1 mediates EGFR-induced cell migration remains elusive. In the present study, we sought to determine whether the lipase activity of PLC-gamma1 is required for EGFR-induced cell migration. We found that overexpression of PLC-gamma1 in squamous cell carcinoma SCC4 cells markedly enhanced EGF-induced PLC-gamma1 activation, intracellular calcium rise, and cell migration. This enhancement was abolished by mutational inactivation of the catalytic domain of PLC-gamma1. Inhibition of the downstream signaling processes mediated by the activity of phospholipase C (PLC) using IP(3) receptor inhibitor or intracellular calcium chelator blocked EGF-induced cell migration. These data indicate that EGF-induced cell migration is mediated by the lipase domain of PLC-gamma1 and the subsequent IP(3) generation and intracellular calcium mobilization.

The vitamin D receptor (VDR) and its ligand 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are required for normal keratinocyte differentiation. Both the epidermis and the hair follicle are disrupted in VDR-null mice. Hairless (Hr), a presumptive transcription factor with no known ligand, when mutated, disrupts hair follicle cycling similar to the effects of VDR mutations. Hr, like VDR, is found in the nuclei of keratinocytes in both epidermis and hair follicle. To investigate the potential interaction between Hr and VDR on keratinocyte differentiation, we examined the effect of Hr expression on vitamin D-responsive genes in normal human keratinocytes. Inhibition of Hr expression in keratinocytes potentiated the induction of vitamin D-responsive genes, including involucrin, transglutaminase, phospholipase C-γ1, and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by 1,25(OH)2D3. Overexpression of Hr in human keratinocytes suppressed the induction of these vitamin D-responsive genes by 1,25(OH)2D3. Coimmunoprecipitation, DNA mobility shift assays, and chromatin immunoprecipitation revealed that Hr binds to VDR in human keratinocytes. Hr binding to the VDR was eliminated by 1,25(OH)2D3, which recruited the coactivator vitamin D receptor-interacting protein 205 (DRIP205) to the VDR/vitamin D response element complex. These data indicate that Hr functions as a corepressor of VDR to block 1,25(OH)2D3 action on keratinocytes.

Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity. In this study, we have investigated the mechanism by which TGF-beta(1), TGF-beta(2) and TGF-beta(3) stimulate biglycan mRNA expression in the osteoblastic cell line MG-63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5' DNA was found to respond to TGF-beta(1) with increased transcriptional activity in a dose-dependent manner. Also TGF-beta(2) and TGF-beta(3), two structurally highly related TGF-beta isoforms stimulated biglycan transcription. A TGF-beta responsive region was identified within the first 218 bp of the human biglycan promoter upstream from the transcriptional start site, which contained several binding sites for the transcription factor Sp1. Electrophoretic mobility shift assays with nuclear extracts from MG-63 cells showed binding of both Sp1 and Sp3 to a site at -216 to -208. When the biglycan promoter construct was co-transfected with Sp1 and Sp3 expression vectors in Sp1-deficient Drosophila Schneider-2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF-beta in MG-63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first time that TGF-beta(1) stimulation of human biglycan mRNA expression relies on increased transcription of the biglycan gene, and is mediated by members of the Sp1 family of transcription factors.

1,25(OH)2D regulates a number of cellular events which contribute to its ability to stimulate differentiation of the keratinocyte. 1,25(OH)2D raises the intracellular calcium (Cai) level in part by increasing the expression of the calcium receptor (CaR). This sensitizes the cell to extracellular calcium, triggering the signaling pathway coupled to the CaR, which results in a rise in Cai. 1,25(OH)2D induces the family of phospholipases C (PLC). These enzymes mediate the hydrolysis of phosphatidyl inositol bisphosphate (PIP2) to form inositol tris phosphate (IP3) and diacylglycerol (DG), which stimulate calcium release from intracellular stores and activate protein kinases C (PKC), respectively. The CaR and other G protein coupled receptors signal through PLC-beta, whereas tyrosine kinase growth factor receptors such as the EGF receptor signal through PLC-gamma. Calcium and PKC regulate the expression of genes in part by controlling the levels and activity of AP-1 transcription factors. 1,25(OH)2D also directly induces structural genes such as involucrin, a substrate for transglutaminase, which crosslinks it to other substrates to form the cornified envelope. 1,25(OH)2D regulates gene expression by activating the vitamin D receptor (VDR), a transcription factor, which, in combination with the retinoid X receptor (RXR) or retinoid A receptor (RAR), binds to its vitamin D response elements (VDRE) in the promoters of genes whose expression it regulates. The VDR also binds to one of two coactivator complexes, Mediator/DRIP (VDR interacting proteins) or p160/SRC (steroid hormone receptor complex), complexes which link the VDR to the RNA polymerase complex. We have recently discovered that the binding of VDR to these complexes is sequential. Binding to Mediator/DRIP occurs in the undifferentiated keratinocyte, but as the cell differentiates, DRIP(205) (the key protein of the DRIP complex binding to the VDR) levels fall, and p160/SRC binding takes over. We hypothesize that this sequential replacement of Mediator/DRIP by p160/SRC is critical for differentiation. Squamous cell carcinomas (SCC) fail to respond to the prodifferentiating actions of 1,25(OH)2D. These cells have normal levels of VDR and normal binding of VDR to VDREs. However, they fail to down-regulate DRIP(205) such that the p160/SRC complex fails to bind to VDR. This lack of sequential binding of these coactivator complexes to the VDR, we believe, maintains the cell in a state of continued proliferation and blocks the ability of 1,25(OH)2D to induce the expression of genes required for the differentiation process.

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.

Squamous cell carcinomas (SCC) derived from human epidermis fail to differentiate normally under the influence of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 [ despite the presence of the vitamin D receptor. Previous studies from our laboratory showed that phospholipase C-γ1 (PLC-γ1) was upregulated transcriptionally by 1,25(OH) 2 D 3 in normal human keratinocytes, and a vitamin D responsive element (VDRE) in its promoter region has been identified. To examine the inducibility of human PLC-γ1 transcription by 1,25(OH) 2 D 3 and/or retinoic acid in SCC cell lines, we transiently transfected SCC4 and SCC12B2 cells with human PLC-γ1 promoter-luciferase constructs containing the VDRE and tested the response of these constructs to 1,25(OH) 2 D 3 and/or all-trans retinoic acid. The induction of the human PLC-γ1 VDRE by 1,25(OH) 2 D 3 was synergistic with all-trans retinoic acid in normal human keratinocytes, but none of the constructs was induced by 1,25(OH) 2 D 3 and/or all-trans retinoic acid in SCC4 and SCC12B2 cells. In contrast, the construct containing the VDRE of the human 24-hydroxylase gene was induced several fold by 1,25(OH) 2 D 3 in normal human keratinocytes and by both 1,25(OH) 2 D 3 and all-trans retinoic acid in SCC4 and SCC12B2 cells. DNA mobility shift assays showed that both the vitamin D receptor and the retinoic acid receptor in SCC4 and SCC12B2 cells bound the human PLC-γ1 VDRE similarly to that seen in normal keratinocytes. The data indicate that the VDRE in the human PLC-γ1 gene is not functional in SCC4 and SCC12B2 cells, unlike normal human keratinocytes, even though vitamin D receptors bind normally to it. Failure of transcriptional control of the PLC-γ1 gene by 1,25(OH) 2 D 3 suggests the lack of a cofactor(s) linking the VDRE to the transcriptional machinery.