Promoting Polarization and Differentiation of Primary Human Salivary Gland Stem/Progenitor Cells in Protease-Degradable Hydrogels via ROCK InhibitionMetkari, Witt, Cognetti
et alACS Appl Mater Interfaces (2025) 17 (12), 18083-18095
Abstract: Toward the goal of in vitro engineering of functional salivary gland tissues, we cultured primary human salivary stem/progenitor cells (hS/PCs) in hyaluronic acid-based matrices with varying percentages of proteolytically degradable crosslinks in the presence of Rho kinase (ROCK) inhibitor. Single cells encapsulated in the hydrogel grew into organized multicellular structures by day 15, and over 60% of the structures developed in the nondegradable and 50% degradable hydrogels contained a central lumen. Importantly, ROCK inhibition led to the establishment of multicellular structures that were correctly polarized, as evidenced by apical localization of a Golgi marker GM130, apical/lateral localization of tight junction protein zonula occludens-1 (ZO-1), and basal localization of integrin β1 and basement membrane proteins laminin α1 and collagen IV. Cultures maintained in 50% degradable gels with ROCK inhibition exhibited an increased expression of acinar markers aquaporin 5 (AQP5, AQP5) and sodium-potassium-chloride cotransporter 1 (SLC12A2, NKCC1) at the transcript and the protein levels, respectively, as compared to those without ROCK inhibition. Upon stimulation with isoproterenol, α-amylase secretion into the lumen was observed. Particle-tracking microrheology was employed to analyze the stiffness of cells using mitochondria as the passive tracer particles. Our results indicated that cells grown in 100% degradable gels were stiffer than those maintained in nondegradable gels, and cells cultured with the ROCK inhibitor were softer than those maintained without the inhibitor. We conclude that reducing cellular contractility via ROCK inhibition while retaining some degree of matrix confinement promotes the establishment of multicellular structures containing pro-acinar cells with correct apicobasal polarization.
Thrombospondin 1 Promotes Cytoskeleton Remodeling, Dedifferentiation, and Pulmonary Metastasis through ITGA1 and ITGA6 in OsteosarcomaXu, Huang, Zhu
et alInt J Biol Sci (2025) 21 (5), 2083-2100
Abstract: Dedifferentiation of osteosarcoma cells leads to poor prognosis. We plan to identify the key molecules that are involved in cell dedifferentiation and explore how they promote the pulmonary metastasis of osteosarcoma cells. We performed a sphere formation assay and confirmed that the spheroid cells could be redifferentiated into osteoblasts, adipocytes, and chondrocytes in specific medium, and the stem cell-like markers Stro-1 and CD117 were detected on the cell surface, which indicated that the spheroid cells were dedifferentiated cells. Thrombospondin 1 (THBS1) and ITGAs were identified as the key molecules in dedifferentiation through mRNA-seq and analysis, and osteosarcoma patients with higher THBS1 expression had a worse prognosis than those with lower THBS1 expression. THBS1 promotes the accumulation of ITGA1 and ITGA6 on the cell membrane in the early phase of dedifferentiation, thereby increasing the phosphorylation of FAK, RasGRF1, and MLC2 in the cytoplasm and promoting cytoskeleton remodeling. Our results suggest that THBS1 promotes cell dedifferentiation and pulmonary metastasis by promoting cytoskeletal remodeling and that ITGA1 and ITGA6 play important roles in mediating extracellular to intracellular signals; this mediating effect takes place mainly in the early phase of dedifferentiation.© The author(s).
Role of the integrin‑β1/TGF‑β1 signaling pathway in the pathogenesis of pelvic organ prolapse: A study on vaginal wall tissue alterations and molecular dysfunctionKong, Wang, Hao
et alMol Med Rep (2025) 31 (4)
Abstract: Pelvic organ prolapse (POP) is a prevalent condition among middle‑aged and older women, and is associated with the irregular production and breakdown of the extracellular matrix. Mechanical forces serve a key role in preserving the equilibrium between matrix synthesis and degradation, thereby supporting the structural integrity of pelvic floor tissues. The aim of the present study was to investigate alterations in the composition of vaginal wall tissues in individuals suffering from POP and to investigate the molecular mechanisms through which mechanical forces trigger fibroblast apoptosis and influence collagen expression via the integrin‑β1/TGF‑β1 signaling pathway. Masson's trichrome and Elastica van Gieson staining were used to examine the pathological alterations in the tissue associated with POP. Analysis of immunofluorescence, western blotting and reverse transcription‑quantitative PCR data was performed to assess changes in the levels of proteins and genes such as collagen, integrin‑β1, TGF‑β1, MMP‑1 and tissue inhibitor of metalloproteinase‑1 (TIMP‑1). Fibroblasts were incubated with an integrin‑β1 antagonist RGD peptide to mimic cellular injury induced by mechanical forces, and cell migration and apoptosis were analyzed using scratch assays and flow cytometry. Cytoskeletal alterations were detected via immunofluorescence staining, and western blot analysis was conducted to examine the expression levels of integrin‑β1, TGF‑β1, TIMP‑1, MMP‑1, collagen type I α1 chain (COL1A1) and collagen type III α1 chain (COL3A1) across various groups. Analysis revealed that in the POP group, the collagen fibers in the vaginal wall tissues were loose and irregularly arranged, the number of elastic fibers was reduced and the structure was degraded. Furthermore, stress fibers were incomplete and their functions were impaired, resulting in damage to the connective tissue structure of the pelvic floor. Integrin‑β1 was key for fibroblast migration, apoptosis and collagen synthesis. Additionally, the integrin‑β1/TGF‑β1 signaling pathway served a role in mediating fibroblast apoptosis, and influencing the synthesis and metabolism of COL1A1 and COL3A1 induced by mechanical forces. Understanding the underlying pathogenesis of pelvic floor organ prolapse could pave the way for future investigations into innovative prevention and treatment strategies.
Recombinant human collagen XVII protects skin basement membrane integrity by inhibiting the MAPK and Wnt signaling pathwaysWang, Lin, Wei
et alMol Med Rep (2025) 31 (4)
Abstract: Collagen XVII is a key component linking the cytoskeleton to the basement membrane, serving an essential role in maintaining skin integrity. With the advancement of synthetic biology, recombinant human collagen XVII (RHCXVII) has emerged as a promising novel collagen material. The present study aimed to elucidate the efficacy and mechanisms of action of RHCXVII in protecting skin basement membrane integrity. A skin injury model was established using ultraviolet B (UVB) irradiation on human HaCaT keratinocytes treated with RHCXVII. The effects of RHCXVII on cell migration and adhesion were assessed using wound healing assay and hematoxylin and eosin staining, respectively. The expression of key extracellular matrix (ECM) components such as collagen IV, collagen VII, laminin 332 and integrin α6 (ITGA6) were quantified using reverse transcription‑quantitative PCR and western blotting. The mechanism of action of RHCXVII in protecting skin basement membrane integrity was investigated using a phosphorylated‑antibody array and verified by western blotting. RHCXVII significantly increased the migration and adhesion of UVB‑irradiated HaCaT cells (P<0.01). Additionally, RHCXVII significantly upregulated expression levels of collagen type IV α1 chain, collagen type VII α1 chain, laminin subunit β3 and ITGA6 in UVB‑irradiated HaCaT cells (P<0.05). RHCXVII significantly inhibited the phosphorylation of p38 and c‑Jun in the MAPK and Wnt signaling pathways (P<0.01). In conclusion, RHCXVII protected skin basement membrane integrity by enhancing migration and adhesion of keratinocytes, upregulating key ECM components and inhibiting protein phosphorylation in MAPK and Wnt pathways. The present study enhanced the current understanding of RHCXVII as a protector of skin basement membrane integrity. Furthermore, the present study highlighted clinical implications and the broad therapeutic potential of RHCXVII in both medical and cosmetic application.
膜杰作
Star Staining
