Glucocorticoid-induced Osteoporosis Research Articles

The pivotal role of the Hes1/Piezo1 pathway in the pathophysiology of glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis (GIOP) lacks fully effective treatments. This study investigated the role of Piezo1, a mechanosensitive ion channel component 1, in GIOP. We found reduced Piezo1 expression in cortical bone osteocytes from patients with GIOP and a GIOP mouse model. Yoda1, a Piezo1 agonist, enhanced the mechanical stress response and bone mass and strength, which were diminished by dexamethasone (DEX) administration in GIOP mice. RNA-seq revealed that Yoda1 elevated Piezo1 expression by activating the key transcription factor Hes1, followed by enhanced CaM kinase II and Akt phosphorylation in osteocytes. This improved the lacuno-canalicular network and reduced sclerostin production and the receptor activator of NF-κB/osteoprotegerin ratio, which were mitigated by DEX. Comparative analysis of mouse models and human GIOP cortical bone revealed downregulation of mechanostimulated osteogenic factors, such as osteocrin, and cartilage differentiation markers in osteoprogenitor cells. In human periosteum-derived cells, DEX suppressed differentiation into osteoblasts, but Yoda1 rescued this effect. Our findings suggest that reduced Piezo1 expression and activity in osteocytes and periosteal cells contribute to GIOP, and Yoda1 may offer a novel therapeutic approach by restoring mechanosensitivity.

Examining osteoprotective properties of gluten-free bread made with a combination of amaranth and tigernut flours

AbstractGluten-free bread (GFB) consisting of amaranth flour, tigernut (chufa tuber) flour, apple powder, carrot powder, and soy protein isolate can have osteoprotective effects. Glucocorticoid-induced osteoporosis (GIO) was chosen as a model of experimental osteoporosis. Experimental studies were carried out on white male Wistar rats (n = 150): Group 1 – healthy animals receiving standard diet (StD); Group 2 – GIO rats receiving StD; Group 3 – GIO animals receiving zoledronic acid (ZA) and StD; Group 4 – GIO rats receiving ZA and GFB; Group 5 – GIO animals receiving GFB. We evaluated the general condition of animals and carried out morphological and biochemical studies. Destructive changes in the spatial structure of the bone tissue in GIO, observed in Group 2, led to the inability of the bone to withstand the functional load. At the same time, isolated treatment with ZA had virtually no effect on the final morphological picture. Including GFB in the diet of experimental animals both alone and with ZA had a noticeable protective effect on conditions triggering GIO.

Prevention and treatment of glucocorticoid-induced osteoporosis in adults: recommendations from the European Calcified Tissue Society.

This report presents the recommendations of the European Calcified Tissue Society (ECTS) for the prevention and treatment of glucocorticoid-induced osteoporosis (GIOP) in adults. Our starting point was that the recommendations be evidence based, focused on non-bone specialists who treat patients with glucocorticoid (GC) and broadly supported by ECTS. The recommendations were developed by global experts. After a comprehensive review of the literature, 25 recommendations were formulated, based on quality evidence. For stratifying fracture risk and the most appropriate first line of treatment, we have classified patients into 3 categories: those at medium risk of fractures, ie, adults without a recent (in the last 2 years) history of fracture; those at high risk of fractures, ie, adults with recent history of fracture, and/or at least one vertebral fracture (grade ≥ 2 according to Genant classification); and those at very high risk of fractures, ie, adults aged ≥70 years with a recent hip fracture, pelvis fracture, and/or at least one vertebral fracture (grade ≥ 2 according to Genant classification). The subtopics in the recommendations include who to assess, how to assess, who to treat, how to treat, and follow-up and monitoring. General measures are recommended for all patients who are being prescribed GCs for ≥3 months, ie, calcium and protein intake should be normalized, a 25(OH) vitamin D concentration of 50-125 nmol/L should be attained, and the risk of falls be minimized. (1) Who to assess? (R1-2) A preliminary assessment of fracture risk should be routinely performed in patients likely to receive oral GCs for ≥3 months: (i) women and men ≥ 50 years and (ii) patients at increased risk of fracture (history of fragility fracture and/or have comorbidities or are on medications that are frequently associated with osteoporosis. (2) How to assess (fracture risk)? (R3-6) Clinical risk factors include history of fragility fracture, systematic vertebral imaging, and GC dose-adjusted FRAX, measurement of bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA), fall risk, and biochemical testing. (3) Who to treat? (R7-12) Anti-osteoporosis treatment is indicated for women and men ≥ 50 years with (i) the presence of a recent history of vertebral and/or non-vertebral fracture (less than 2 years), (ii) and/or a GC dosage ≥ 7.5 mg/day, (iii) and/or age ≥ 70 years, (iv) and/or a T-score ≤ -1.5, (v) and/or 10-year probability risk above the country specific GC dose-adjusted FRAX® thresholds. In premenopausal women and men < 50 years with a Z-score ≤ -2 and/or a history of fragility fracture, it is recommended to refer the patient to a bone specialist. (4) How to treat? (R13-18) In women and men ≥ 50 years, (i) alendronate or risedronate is preferred as the first line of treatment in patients at medium risk of fractures, (ii) zoledronic acid or denosumab in patients at high risk of fractures, and (iii) teriparatide in patients at very high risk of fractures. It is imperative that sequential therapy be implemented in individuals receiving denosumab or teriparatide as their first-line treatment regimen. (5) Follow-up and monitoring (R19-25): in patients receiving anti-osteoporosis treatment, monitoring of clinical risk factors (eg, history of fragility fracture), systematic vertebral imaging, fall risk, BMD measurement using DXA, and biochemical testing should be performed regularly during follow-up. The new, evidence-based recommendations by the ECTS for the prevention and treatment of GIOP provide clear and pragmatic advice to all health practitioners especially those who are not bone specialists.

The effect of the Hippocampus erectus decoction on improving osteoporosis in zebrafish

The seahorse is a traditional Chinese herbal medicine that has been utilized for the treatment of conditions such as osteoporosis, nonunion fractures, knee osteoarthritis, and herniated lumbar disks.Zebrafish is an excellent animal model to study the effects of herbal preparations on osteogenesis and bone metabolism, and bone biology is highly similar to that of mammals, so it has been used to develop multiple glucocorticoid-induced osteoporosis (GIOP) models. Our study aimed to explore whether the decoction of Hippocampus erectus (DHe) administered in zebrafish larvae promotes bone health under physiological and osteoporotic conditions. In zebrafish larvae, 25 µM prednisolone solution was successfully used to induce the zebrafish osteoporosis model, which can significantly inhibit the mineralization area and degree of bone mineralization, reduce the activity of alkaline phosphatase, and increase tartrate-resistant acid phosphatase activity. The results showed that 0.3 g/L DHe had a significant improvement effect on osteoporosis in zebrafish by enhancing the mineralization degree and phosphatase activity of zebrafish, while also promoting the expression of genes associated with bone development. It can be considered a good candidate to develop new drugs against osteoporosis.

Generation of BT-Amide, a Bone-Targeted Pyk2 Inhibitor, Effective via Oral Administration, for the Prevention of Glucocorticoid-Induced Bone Loss.

Glucocorticoid-induced osteoporosis (GIOP) is the leading cause of iatrogenic osteoporosis due to the widespread clinical use of glucocorticoids (GC) as immunosuppressants. Previous research identified the proline-rich tyrosine kinase 2, Pyk2, as a critical mediator of GC-induced bone loss, and that blocking Pyk2 could protect the skeleton from adverse GC actions. However, systemic administration of current Pyk2 inhibitors causes harmful side effects, such as skin lesions. To address this, we developed bone-targeted (BT) Pyk2 inhibitors by conjugating them with bisphosphonates (BP), ensuring adherence to the bone matrix and reducing impact on noncalcified tissues. We synthesized BT-Amide by linking a derivative of TAE-226, a Pyk2 inhibitor, with alendronic acid. Oral administration (gavage) of BT-Amide prevented GC-induced bone loss in mice without causing skin lesions, or elevation of any organ toxicity markers. These findings introduce BT-Amide as the first orally effective bone-targeted Pyk2 inhibitor for preventing GC-induced bone loss while minimizing off-target effects.

Rehmannioside A promotes the osteoblastic differentiation of MC3T3-E1 cells via the PI3K/AKT signaling pathway and inhibits glucocorticoid-induced bone loss in vivo

Glucocorticoid-induced osteoporosis (GIOP) is a widespread disease characterized by low bone density. There remains a lack of effective means for osteoporosis. Rehmannioside A (ReA), an iridoid glycoside, exhibits various pharmacological activities. This study aimed to explore the role and mechanism of ReA in osteogenic differentiation of osteoblasts. Cell viability, reactive oxygen species (ROS) generation, and cell apoptosis were assessed using corresponding assay kits. Real-time quantitative polymerase chain reaction, Western blotting, and alkaline phosphatase (ALP) staining were performed to evaluate the osteogenic differentiation of MC3T3-E1 cells. Alizarin red S staining was used to assess the mineralization of MC3T3-E1 cells. Protein expression associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway was analyzed using Western blotting. Micro-computed tomography, histopathological, and immunohistochemical analyses were performed to determine the therapeutic effect of ReA on GIOP in vivo.The results showed that ReA promoted the osteogenic differentiation of MC3T3-E1 cells by regulating the PI3K/AKT signaling pathway and protected mice against glucocorticoid-induced bone loss by promoting osteoblast-mediated bone formation in vivo. The findings of the current study revealed that ReA is a potential therapeutic agent for osteoporosis.

Apoptosis-associated genetic mechanisms in the transition from rheumatoid arthritis to osteoporosis: A bioinformatics and functional analysis approach.

This study explores the mechanisms of glucocorticoid-induced osteoporosis (OP) and Rheumatoid arthritis (RA), focusing on apoptosis and its role in the progression from RA to OP. Using microarray data from the GEO database, differential gene expression analysis was conducted with the limma package, identifying significant genes in RA and OP. Weighted Gene Co-expression Network Analysis (WGCNA) further examined gene relationships with the disease status, identifying co-expression patterns. Key genes were pinpointed by intersecting differentially expressed genes from RA and OP datasets with WGCNA module genes. Functional enrichment analysis using the "clusterProfiler" package focused on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Machine learning methods, including Lasso and Random Forest, refined the selection of key genes related to apoptosis. Immune infiltration analysis using CIBERSORT assessed immune cell differences between disease and normal samples. The study highlighted two crucial genes: ATXN2L and MMP14. These genes were identified through various analyses and found to be significantly associated with the progression of RA and OP. Gene Set Enrichment Analysis of ATXN2L and MMP14 revealed their involvement in specific biological processes and pathways. Correlation analysis between these key genes and immune cell infiltration showed significant associations. The ROC analysis evaluated the diagnostic performance of ATXN2L and MMP14, with miRNA regulatory networks related to these genes also predicted. In summary, this research provides valuable insights into the molecular mechanisms of RA and OP, emphasizing the importance of apoptosis and immune processes.

Denosumab Response in Glucocorticoid-Induced Osteoporosis Resistant to Bisphosphonate Therapy in an Adolescent with Childhood Onset Minimal Change Disease

Glucocorticoid induced osteoporosis is difficult to treat in patients with nephrotic syndrome. The efficacy and safety of denosumab in pediatric population is unknown. We report the successful use of denosumab in a child who was non responsive to conventional measures and bisphosphonates.

The Putative Role of Nigella Sativa Oil versus Calcium and Vitamin D in Modulating the Glucocorticoid-Induced Osteoporosis in Rats: Histological and Immunohistochemical Study.

The Putative Role of Nigella Sativa Oil versus Calcium and Vitamin D in Modulating the Glucocorticoid-Induced Osteoporosis in Rats: Histological and Immunohistochemical Study.

A size-switchable microsphere loaded with salmon calcitonin as two-weekly dosing for osteoporosis therapy

Osteoporosis is a disease with an increased incidence of fractures due to decreased bone mass and destruction of the microstructure of bone tissue. Salmon calcitonin (sCT), as a peptide, possesses the ability to inhibit osteoclast activity and thus regulate bone metabolism in clinical. However, short half-life and unstable physicochemical properties leading to rapid degradation of sCT have severely limited its clinical application. In this study, a size-switchable microsphere was developed to solve the problem of frequent administration and poor stability of sCT. sCT was encapsulated into Egg PC to form anhydrous reverse micelles (ARM) and then ARM was encapsulated into microspheres (MS@ARM). The degradable composite microspheres were utilized to provide a drug reservoir for sustained release of ARM encapsulated with sCT to reduce the frequency of drug administration, while the released ARM encapsulated with sCT entered the blood circulation to further protect sCT. In vitro release experiments demonstrated that the microspheres could sustain the release of sCT for at least 16 days. The microspheres MS@ARM showed the advanced therapeutic effect on the mouse model of glucocorticoid-induced osteoporosis (GIOP) at a low dosing frequency. The size-switchable microsphere is expected to be a new strategy for delivering sCT for osteoporosis treatment.

Soy Peptide Ameliorate TGF-β1-Mediated Osteoblast Differentiation through Smad and MAPK Signaling Pathways.

The aim of this study was to determine the osteogenic activity and mechanism of soybean peptide VVELLKAFEEKF (SOP) and the potential relationship between SOP and transforming growth factor-β1 (TGF-β1). The results show that SOP promotes MC3T3-E1 cell proliferation by altering cell progression. SOP induced cell differentiation and mineralization in a dose-dependent manner at 0.7-7 μM. Moreover, SOP stimulates osteoblast differentiation, which may be achieved through the activation of p38-MAPK and Smad2/3 signaling pathways. Furthermore, treatment with a TβRI inhibitor (SB525334) inhibited the phosphorylation levels of p38 and Smad2/3, which indicates the involvement of TβRI in the process of osteoblast differentiation caused by SOP. Besides, in non-FBS-cultured MC3T3-E1 cells, SOP and TGF-β1 promoted the phosphorylation of Smad2/3 and alkaline phosphatase (ALP) activity, but the effect was lost when SOP was incubated separately, indicating that SOP stimulated osteoblast differentiation by promoting TGF-β1 activity. In vivo, SOP significantly restores bone mineral density loss and behavioral deficits in a model of glucocorticoid-induced osteoporosis (GIOP) in zebrafish. These results suggest that SOP may have the function of promoting bone remodeling and may be used as a potential active factor for functional food development to prevent osteoporosis.

The protective effects of tocotrienols and coenzyme Q10 against glucocorticoid-induced osteoporosis in rats: Potential roles of Nrf2, Bcl-2, and autophagy.

The protective effects of tocotrienols and coenzyme Q10 against glucocorticoid-induced osteoporosis in rats: Potential roles of Nrf2, Bcl-2, and autophagy.

Current Treatment for Glucocorticoid-Induced Osteoporosis: Beyond Bisphosphonates

Current Treatment for Glucocorticoid-Induced Osteoporosis: Beyond Bisphosphonates

Antiosteoporosis and bone protective effect of alpinumisoflavone in steroid-induced osteoporosis rats via alteration of apoptosis, inflammatory and RANK/RANKL/OPG signaling pathway

Objective: To estimate the bone protective effect of alpinumisoflavone, a natural prenylated isoflavonoid, against glucocorticoid-induced osteoporosis in rats. Methods: Male Wistar rats received intramuscular administration of dexamethasone (4 mg/mL) at a dose level of 7 mg/kg for 5 weeks, and then alpinumisoflavone (5, 10, and 15 mg/kg) and alendronate (2 mg/kg) from 2 weeks. The body weight and organ weight (femoral, vagina, and uterus) were estimated. MicroCT analysis, bone turnover markers, bone parameters, oxidative stress parameters, and inflammatory cytokines were estimated. mRNA expressions of related genes were also estimated. Results: Alpinumisoflavone remarkably boosted body weight and organ weight (ureters and vagina), improved microCT analysis parameters, and boosted levels of bone markers. Besides, alpinumisoflavone considerably (P&lt;0.001) restored the level of bone turnover markers and oxidative stress parameters, remarkably suppressed the level of cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor-α, and increased transforming growth factor-β and insulin-like growth factor. It also significantly restored the osteoprotegerin (OPG, RANKL, and OPG/RANKL ratio) levels and the mRNA expression of Caspase (3, 6, 7, 9), BMPs, OPN, ALP, RUNX2, and OCN. Conclusions: The current result suggests the bone protective effect of alpinumisoflavone against glucocorticoid-induced osteoporosis in rats.

Severity of acute phase reaction in children receiving the first dose of zoledronic acid and the impact of the underlying condition: a cross-sectional study

IntroductionZoledronic acid (ZA), used for treatment of children with osteoporosis, can cause acute phase reaction (APR) following the first infusion. Many institutions have a policy to admit and monitor all...

Comparative performance analysis of large language models: ChatGPT-3.5, ChatGPT-4 and Google Gemini in glucocorticoid-induced osteoporosis

BackgroundsThe use of large language models (LLMs) in medicine can help physicians improve the quality and effectiveness of health care by increasing the efficiency of medical information management, patient care, medical research, and clinical decision-making.MethodsWe collected 34 frequently asked questions about glucocorticoid-induced osteoporosis (GIOP), covering topics related to the disease’s clinical manifestations, pathogenesis, diagnosis, treatment, prevention, and risk factors. We also generated 25 questions based on the 2022 American College of Rheumatology Guideline for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis (2022 ACR-GIOP Guideline). Each question was posed to the LLM (ChatGPT-3.5, ChatGPT-4, and Google Gemini), and three senior orthopedic surgeons independently rated the responses generated by the LLMs. Three senior orthopedic surgeons independently rated the answers based on responses ranging between 1 and 4 points. A total score (TS) > 9 indicated ‘good’ responses, 6 ≤ TS ≤ 9 indicated ‘moderate’ responses, and TS < 6 indicated ‘poor’ responses.ResultsIn response to the general questions related to GIOP and the 2022 ACR-GIOP Guidelines, Google Gemini provided more concise answers than the other LLMs. In terms of pathogenesis, ChatGPT-4 had significantly higher total scores (TSs) than ChatGPT-3.5. The TSs for answering questions related to the 2022 ACR-GIOP Guideline by ChatGPT-4 were significantly higher than those for Google Gemini. ChatGPT-3.5 and ChatGPT-4 had significantly higher self-corrected TSs than pre-corrected TSs, while Google Gemini self-corrected for responses that were not significantly different than before.ConclusionsOur study showed that Google Gemini provides more concise and intuitive responses than ChatGPT-3.5 and ChatGPT-4. ChatGPT-4 performed significantly better than ChatGPT3.5 and Google Gemini in terms of answering general questions about GIOP and the 2022 ACR-GIOP Guidelines. ChatGPT3.5 and ChatGPT-4 self-corrected better than Google Gemini.

CUL1 exacerbates glucocorticoid-induced osteoporosis by enhancing ASAP1 ubiquitination.

Glucocorticoid-induced osteoporosis is a leading secondary cause of osteoporosis. Cullin-1 (CUL1) levels are abnormally elevated in patients with osteoporosis, but the underlying mechanism remains unclear. The purpose of this study was to elucidate the mechanism of action of CUL1 in a glucocorticoid (dexamethasone, Dex)-induced osteoporosis model. C57BL/6J mice were intraperitoneally injected with Dex to establish an osteoporosis model. Mouse femur bone injury and bone formation were detected using hematoxylin-eosin or Masson staining. Apoptosis and cell cycle distribution were determined by flow cytometry. Alkaline phosphatase (ALP) activity and calcified nodules were monitored using ALP and Alizarin Red S staining. The molecular mechanism was validated by co-immunoprecipitation (Co-IP) and ubiquitination assays. CUL1 expression was enhanced in the Dex-induced osteoporosis mouse model. CUL1 silencing moderated the Dex-induced cell proliferation and osteogenesis inhibition. Moreover, CUL1 promoted the ubiquitination and degradation of ASAP1 via the SKP1-CUL1-F-box (SCF)-FBXW7 complex. CUL1 induced apoptosis and repressed osteogenesis by ASAP1. CUL1 silencing alleviated the Dex-induced osteoporosis in mice. CUL1 suppressed osteoblast proliferation and osteogenesis by promoting ASAP1 ubiquitination via the SCF-FBXW7 complex in glucocorticoid-induced osteoporosis.

Treatment of glucocorticoid-induced osteoporosis with concurrent denosumab and romosozumab: a case report.

Osteoporosis is a metabolic bone disorder for which treatment options include antiresorptive therapies (e.g., bisphosphonates, denosumab); anabolics (e.g., teriparatide, abaloparatide); and dual mechanisms (e.g., romosozumab). Management of osteoporosis with concurrent antiresorptive and anabolic agents may be superior to monotherapy, as demonstrated in the DATA trial with the combination of denosumab and teriparatide. However, there is limited experience with the combination of denosumab and romosozumab, which may be an alternative antiresorptive/anabolic regimen for individuals who are not candidates for PTH receptor agonists. In this case, we present a young man with glucocorticoid-induced osteoporosis who could not tolerate a daily injectable anabolic and who experienced improvement in bone mineral density with concurrent denosumab and off-label romosozumab administration.

Ukrainian guidelines for the prevention and treatment of glucocorticoid-induced osteoporosis

Introduction. Glucocorticoid-induced osteoporosis (GIOP) is a bone metabolic disease that develops as a result of glucocorticoid (GC) use, is one of the most frequent causes of secondary osteoporosis, leads to an increase in the risk of low-energy fractures, disability and mortality of GC users. Despite the few stu­dies conducted in Ukraine on the epidemiology and treatment of GIOP, there have been no national recommendations for its ma­nagement until now, which has become the background for their creation. The purpose was to develop national guidelines for the diagnosis, prevention, and treatment of GIOP based on the analytical analysis of modern literary sources to improve the awareness of the medical community of Ukraine, improve management, and reduce the socio-economic burden of the disease. Materials and ­methods. A group of 18 experts, leading Ukrainian scientists in various fields, was created to develop the guideline. An analysis of current literary sources on epidemiology, risk factors, diagnosis, prevention and treatment of GIOP, and monitoring of the effectiveness and safety of its treatment was carried out. Evidence was synthesized ­using the GRADE system, and a critical assessment of the quality of the recommendations was carried out using the AGREE II tool. ­Results. The guideline consists of 12 provisions on screening, diagnosis, prevention, and treatment of GIOP. The authors emphasized the need to increase the awareness of the medical community of Ukraine regarding the problem, as well as patients regarding the possible side effects of GC. They presented the features of the clinical assessment of the osteoporotic fracture risk in GC users, as well as modern and available in Ukraine methods of diagnosis and treatment of GIOP. Conclusion. The first Ukrainian guideline for screening, diagnosis, prevention, and treatment of GIOP is an important national tool for its management, which is recommended by the Board of the Ukrainian Association of Osteoporosis for use in practical health care by doctors of various specialties.

Mechanism of modified danggui buxue decoction in glucocorticoid-induced osteoporosis: A discussion based on network pharmacology and molecular docking

ObjectiveGlucocorticoid-induced osteoporosis (GIOP) represents a major complication arising from the long-term use of glucocorticoids, which are widely prescribed for various inflammatory and autoimmune conditions. Despite its prevalence, the current therapeutic options for GIOP are limited in terms of efficacy, safety profiles, and patient compliance. The Modified Danggui Buxue Decoction (DGBXD), a traditional Chinese herbal formulation, has shown promise in preliminary studies for its potential osteoprotective effects. The present study aimed to explore the mechanistic underpinnings of DGBXD's action on GIOP using network pharmacology and molecular docking approaches, bridging traditional medicine with modern pharmacological insights. MethodNetwork pharmacology is applied to screen drug-active compounds and potential core target proteins for disease treatment and to explore the drugs’ therapeutic mechanisms. ResultAltogether, 78 DGBXD active compounds and 223 DGBXD-related, 146 component-disease common, and 2168 GIOP-associated target genes were obtained. The PPI network had 43 nodes and 462 edges, and a total of 10 core target genes, including TP53, JUN and MAPK3, were identified. The results of the GO enrichment analysis implied that DGBXD might participate in biological activities, including responses to oxidative stress and nutrient levels. The outcomes of the KEGG pathway enrichment analysis showed that DGBXD may treat GIOP through TNF, IL-17, and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways. Based on to the molecular docking results, biologically active compounds (beta-carotene, formononetin, luteolin, and isorhamnetin) exhibited good binding to AKT1 and ESR1. ConclusionDGBXD may aid in GIOP treatment by modulating multiple therapeutic targets and signaling pathways.