Fibroblast Activation Protein-α Interacts with CXCL12 to Inactivate Canonical Wnt Signaling and Regulate Osteoblast Differentiation

Endocrinology Geriatrics Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine Orthopedics
fibroblast activation protein-α CXCL12 Wnt/β-catenin pathway osteoblast differentiation adipocyte differentiation osteoporosis mesenchymal progenitor cells

Academic Research Background

With the global population aging rapidly, osteoporosis has gradually become a major disease threatening public health. Characterized by reduced bone mass and deterioration of bone microarchitecture, osteoporosis significantly increases the risk of fractures, severely impacts the quality of life of the elderly, and brings enormous medical burdens. The primary cause of osteoporosis is the imbalance between bone resorption and bone formation. Although current research on bone resorption mechanisms is extensive and related drugs have been successively approved, understanding of bone formation disorders—especially the mechanisms regulating osteoblast differentiation—remains inadequate, limiting the development of new therapies. Therefore, elucidating the cellular and molecular mechanisms governing osteoblast differentiation is crucial for clarifying the pathogenesis of bone metabolic diseases and discovering new intervention targets.

Bone marrow stromal cells (BMSCs) are progenitors of osteoblasts, adipocytes, and other cell types. Under the regulation of specific microenvironments and cell signaling, they can differentiate into osteoblasts for new bone formation or transform into adipocytes. The differentiation fate of BMSCs depends not only on the precise regulation of multiple signaling pathways (such as the Wnt/β-catenin pathway, RUNX2, Osterix, PPARγ, etc.), but also on the imbalance of their differentiation tendency, which is a key factor in diseases like osteoporosis—for instance, increased differentiation towards adipocytes inhibits osteogenesis, increases bone marrow fat content, and indirectly weakens bone tissue function. The specific molecular mechanisms underlying this process remain unclear, especially regarding how the interplay between signaling networks determines cell fate.

In recent years, fibroblast activation protein-α (FAPα), a cell surface serine protease, has gradually been recognized as closely related to bone metabolism. Preliminary reports indicate that FAP possesses dual enzymatic activities (dipeptidyl peptidase and endopeptidase), participates not only in tissue repair, tumors, and inflammation, but also possibly in the negative regulation of osteogenesis. However, whether FAP directly regulates osteogenic and adipogenic differentiation of bone marrow stromal cells, and what molecular targets and pathways are involved in this process, still lack systematic and direct evidence.

Article Source and Basic Information

The paper titled “Fibroblast activation protein- interacts with cxcl12 to inactivate canonical wnt signaling and regulate osteoblast differentiation” was authored by Yuan Dong, Xingli Hu, Wei Liu, Yinglong Hao, Jie Zhou, Xiaoxia Li, and Baoli Wang, from the Chu Hsien-I Memorial Hospital & Institute of Endocrinology and the College of Basic Medical Sciences at Tianjin Medical University. The article was published in the journal Stem Cells (Oxford University Press; doi:10.1093/stmcls/sxaf027) and appeared as an advance article in June 2025.

Overall Research Design and Experimental Methodology Details

This study utilized “mouse bone marrow stromal cells and osteoprogenitor cells” as models and systematically explored the molecular mechanism of FAP in BMSC differentiation by integrating molecular cloning, gene interference (siRNA/shRNA), overexpression, co-immunoprecipitation, protein degradation, functional differentiation assays (osteogenic and adipogenic induction and staining), immunoblotting (Western blot), and transcriptome sequencing (RNA-seq), among various other experimental approaches. The main experimental procedures included:

1. Cell Acquisition and Differentiation Model Establishment

  • Subjects and Samples: BMSCs were isolated from 3-week-old C57BL/6J mice, and osteoprogenitor cell line MC3T3-E1, mesenchymal cell line C3H10T1/2, and murine BMSC line St2 were used.
  • Differentiation Induction: BMSCs, St2, and C3H10T1/2 cells were induced to differentiate into osteoblasts and adipocytes by osteogenic and adipogenic agents, respectively.
  • Differentiation Assessment: Alkaline phosphatase (ALP) staining and activity assays, Alizarin Red staining for mineralization, and Oil Red O staining for adipocyte differentiation were performed.

2. FAP Functional Manipulation (Gain-of-function / Loss-of-function Studies)

  • Overexpression Experiments: FAP gene was cloned into a pcDNA3.1(+) vector, and cells including St2, MC3T3-E1, and C3H10T1/2 were transfected using polyamine transfection reagents to establish FAP-overexpressing models.
  • Gene Knockdown Experiments: Specific siRNA/shRNA were synthesized and transfected or used to infect BMSCs via lentivirus to achieve FAP knockdown.
  • Detection System: The impact of FAP expression on BMSC osteogenic/adipogenic differentiation was compared, with qPCR and Western blot detecting expression of markers such as RUNX2, Osterix, ALP, Osteopontin, PPARγ, C/EBPα, FABP4, and Adipsin.

3. Exploring Molecular Mechanisms

  • Bioinformatics and Co-immunoprecipitation: The STRING database predicted that CXCL12 (C-X-C motif chemokine ligand 12) was an important FAP interactor. Co-immunoprecipitation (Co-IP) then confirmed a direct binding between FAP and CXCL12 proteins.
  • Protein Degradation Assays: In vitro recombinant protein systems were used to analyze whether FAP could directly cleave and degrade CXCL12 and to evaluate the efficiency and time-dependency of this process.
  • Identification of Regulation Level: After FAP overexpression or knockdown, changes in CXCL12 protein and mRNA levels were assessed to determine that the regulation occurred primarily at the protein level.

4. Wnt/β-catenin Signaling Pathway Analysis

  • Monitoring Signaling Activity: Expression levels of key Wnt pathway components (phosphorylated LRP6, phosphorylated GSK3β, non-phosphorylated β-catenin, TCF7L2, etc.) were measured to clarify how FAP and CXCL12 regulated the Wnt/β-catenin pathway and the differentiation fate.
  • Chemical Inhibitor/Activator and Double-gene Manipulation: GSK3β inhibitor CHIR99021 was used to activate β-catenin, and β-catenin plus FAP/CXCL12 double siRNA/expression plasmids were deployed to validate the direct impact of FAP/CXCL12 on Wnt/β-catenin signaling and downstream differentiation outcomes.

5. Exploration of FAP Protease Activity

  • Active-site Point Mutation: FAP was mutated at S624A to generate a protease-dead FAP (FSM). The differences between FSM and wild-type FAP in CXCL12 hydrolysis and differentiation regulation were compared to determine the necessity of enzymatic activity in this regulatory axis.

6. RNA-seq Whole-transcriptome Sequencing

  • Experimental Design: St2 cells with FAP or CXCL12 silenced by siRNA were subjected to whole-transcriptome sequencing to analyze common and differentially regulated gene sets and signaling pathways.
  • Data Analysis: KEGG and GO functional enrichment analyses were conducted to reveal different signaling pathways and molecular network connections, deepening the understanding of the FAP-CXCL12-Wnt regulatory axis.

Details of Major Experimental Results

1. Correlation Between FAP Expression and Differentiation Fate

  • FAP expression was significantly upregulated during osteogenic and adipogenic differentiation of BMSCs, highly expressed in mouse bone and skeletal muscle, and further increased in bones of aged mice, suggesting its possible association with bone metabolic disorders.

2. FAP Influences BMSC Differentiation Fate

  • Osteogenic Differentiation: FAP overexpression markedly inhibited osteogenic differentiation of St2 and MC3T3-E1 cells, as shown by reduced ALP staining, lowered mineralization, and downregulation of osteogenic markers. Conversely, FAP knockdown/silencing promoted osteogenic differentiation.
  • Adipogenic Differentiation: FAP overexpression promoted adipogenic differentiation of C3H10T1/2 cells, enhancing Oil Red O staining and adipocyte marker expression; FAP downregulation inhibited adipogenesis.
  • In primary BMSC validation, FAP knockdown promoted osteogenesis and reduced adipogenesis, confirming its bidirectional regulation.

3. FAP-targeted Degradation of CXCL12 Affects Signaling Pathways

  • FAP forms a complex with CXCL12 protein and can directly cleave and degrade CXCL12 with no effect on its mRNA, clearly acting at the protein level.
  • In purified in vitro recombinant systems, FAP significantly and progressively degraded CXCL12.
  • CXCL12 itself activates canonical Wnt/β-catenin signaling, promoting osteogenesis and inhibiting adipogenesis; FAP protease-dead mutant (FSM) cannot degrade CXCL12 and loses its regulatory effect on BMSC differentiation.

4. FAP, CXCL12, and Wnt/β-catenin Pathway Regulation of Osteogenic and Adipogenic Differentiation

  • FAP upregulation led to the downregulation of Wnt/β-catenin pathway proteins (p-LRP6, p-GSK3β, non-phosphorylated β-catenin, TCF7L2), corresponding to inhibited osteogenesis and promoted adipogenesis; FAP knockdown or CXCL12 overexpression elevated these signals.
  • CHIR99021 activation of β-catenin could reverse the abnormal differentiation caused by FAP, and β-catenin siRNA could attenuate or abolish the regulatory effects of FAP/CXCL12 on differentiation fate, confirming the centrality of this signaling pathway.

5. The Role of CXCL12 in FAP’s Regulation of Differentiation and Cooperation

  • Regulation of CXCL12 expression critically influences BMSC differentiation: its downregulation suppresses osteogenesis and promotes adipogenesis, while upregulation has the opposite effect.
  • CXCL12 siRNA can partially reverse the increased osteogenesis and suppressed adipogenesis caused by FAP siRNA.
  • Conversely, CXCL12 overexpression can partly counteract the inhibited osteogenesis and enhanced adipogenesis induced by FAP overexpression.

6. RNA-seq Reveals Transcriptomic Regulatory Networks

  • More than a thousand differentially expressed genes were detected after silencing FAP or CXCL12 respectively; although the commonly regulated pathways were limited (such as NOD-like receptor, p53, TLR, etc.), KEGG did not identify the Wnt/β-catenin pathway as a major downstream pathway, suggesting the regulation of Wnt by FAP/CXCL12 mainly occurs at the post-transcriptional level.

Research Conclusions and Scientific Value

The study clearly demonstrates that FAP achieves key regulation of osteogenic/adipogenic differentiation fate of mesenchymal progenitor cells by directly cleaving the CXCL12 protein and inhibiting its function of activating Wnt/β-catenin signaling. Specifically, FAP upregulation suppresses Wnt signaling and drives BMSCs to differentiate into adipocytes rather than osteoblasts; reduction of FAP promotes osteogenesis and inhibits adipogenesis. The discovery of this signaling axis offers a new molecular explanation for the reciprocal relationship between bone marrow adiposity and osteogenesis, and provides an important potential target for the intervention of osteoporosis and other skeletal metabolic diseases. Inhibiting FAP expression or activity is expected to serve as a new future strategy for preventing and treating osteoporosis.

Research Highlights

  1. Mechanistic Innovation: For the first time, the study established the complete mechanism by which FAP directly hydrolyzes CXCL12 to regulate Wnt/β-catenin signaling and BMSC differentiation fate, filling the gap in direct evidence for FAP’s involvement in osteogenic regulation.
  2. Methodological Rigor: The research integrates cellular experimentation, in vitro protein reactions, animal tissue detection, differentiation staining, molecular interaction analysis, signaling pathway analysis, and whole-transcriptome sequencing from multiple dimensions, supporting robust conclusions.
  3. Essential Role of Protease Activity: Through site-directed mutation design, it was confirmed that FAP’s enzymatic activity is indispensable for CXCL12 cleavage and functional regulation, providing guidance for the design of specific FAP inhibitors.
  4. Potential Clinical Value: Targeting FAP or CXCL12 may become a new approach to improving the marrow “fat-bone” balance and preventing or reversing osteoporosis.

Other Valuable Content

  • This study also notes that FAP-related pathways and transcriptome changes extend beyond the Wnt pathway, suggesting it may influence bone and fat metabolism on multiple levels.
  • The team previously found that the histone demethylase KDM7A regulates bone homeostasis by controlling FAP transcription, indicating synergistic effects of epigenetic and enzymatic regulation within the bone metabolism axis and broadening the understanding of bone biology networks.

Final Remarks and Significance

This research reveals the bridging role of the new FAP-CXCL12-Wnt/β-catenin signaling axis in fate decisions of bone marrow mesenchymal cell differentiation, providing fresh perspectives for the basic mechanisms, target validation, and drug development related to skeletal metabolic diseases. If the physiological and pathological significance of this axis is further confirmed in animal models and clinical samples in the future, it will greatly enrich the theoretical system of bone biology and advance translational research for osteoporosis and related diseases.