Progression to metastasis is the leading cause of most cancer-related mortality; however, much remains to be understood about what facilitates the spread of tumor cells. In the present study, we describe a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We examined nuclear factor I-C (NFI-C) expression in MCF10A human breast epithelial cells, MCF7 non-invasive breast cancer cells, and MDA-MB231 invasive breast cancer cells by real-time PCR and western blotting. To investigate the loss- and gain-function of NFI-C, we determined whether NFI-C regulated KLF4 expression by real-time PCR, western blotting, and promoter assay. To understand the biological functions of NFI-C, we observed cell invasion, migration, adhesion in human tumor cells by transwell assay, wound healing assay, quantitative RT-PCR, cell adhesion assay, western blotting, and immunohistochemistry. We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT. NFI-C is expressed in normal mammary gland or noninvasive breast cancer cells with epithelial characteristics. NFI-C overexpression induced expression of KLF4 and E-cadherin, but not Slug, in breast cancer cells. NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis. Cells overexpressing NFI-C maintained their epithelial differentiation status, which could drive mesenchymal-epithelial transition (MET) via the NFI-C-KLF4-E-cadherin axis in breast cancer cells. Consequently, NFI-C suppressed EMT, migration, and invasion in breast cancer cells. Our study reveals a novel signaling pathway that is important during breast cancer tumorigenesis: the NFI-C-KLF4-E-cadherin pathway. The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.

We aim to examine the effects of a newly developed peptide derived from CPNE7 (Cpne7-DP) in tertiary dentin formation and peritubular space occlusion, and comprehensively evaluate its potential as a bioactive therapeutic agent. Human dental pulp cells (HDPCs) and a mouse pre-odontoblast cell line, MDPC-23, were chosen for in vitro studies to characterize lineage-specific cell responses after Cpne7-DP treatment. Whether Cpne7-DP reproduces the dentin regenerative potential of CPNE7 was tested using a beagle dog model by generating dentinal defects of various degrees in vivo. Peritubular space occlusion was further examined by scanning electron microscopy and microleakage test, while overall mineralization capacity of Cpne7-DP was tested ex vivo. CPNE7 promotes tubular dentin formation under both shallow and deep dentinal defects, and the functional peptide Cpne7-DP induces odontoblast-like differentiation in vitro, mineralization ex vivo, and tubular dentin formation in in vivo beagle dog dentin exposure and pulp exposure models. Moreover, Cpne7-DP leads to peritubular space occlusion and maintains stability under different conditions. We show that CPNE7 and its derivative functional peptide Cpne7-DP promotes dentin regeneration in dentinal defects of various degrees and that the regenerated hard tissue demonstrates the characteristics of true dentin. Limitations of the current dental materials including post-operative hypersensitivity make biological repair of dentin a field of growing interest. Here, we suggest that the dual functions of Cpne7-DP in tubular dentin formation and peritubular space occlusion are promising for the treatment of dentinal loss and sensitivity.

Tooth development includes numerous cell divisions and cell-cell interactions generating the stem cell niche. After an indefinite number of divisions, pluripotent cells differentiate into various types of cells. Nuclear factor I (NFI) transcription factors are known as crucial regulators in various organ development and stem cell biology. Among its members, nuclear factor I-C (NFI-C) has been reported to play an essential role in odontogenesis. Nfic knockout mice show malformation in all mineralized tissues, but it remains unclear which stage of development Nfic is involved in. We previously reported that Nfic induces the differentiation of ameloblast, odontoblast, and osteoblast. However, the question remains whether Nfic participates in the late stage of development, perpetuating the proliferation of stem cells. This study aimed to elucidate the underlying mechanism of NFI-C function in stem cells capable of forming hard tissues. Here, we demonstrate that Nfic regulates Sox2 and cell proliferation in diverse mineralized tissue stem cells such as dental epithelial stem cells (DESCs), dental pulp stem cells, and bone marrow stem cells, but not in fibroblasts. It was also involved in the expression of pluripotency genes Lin28 and NANOG. Especially in DESCs, Nfic regulates the proliferation of epithelial cells via epithelial-mesenchymal interactions, which are the Fgf8-Nfic-Sox2 pathway in epithelium and Nfic-Fgf10 in the mesenchyme. Moreover, Nfic slightly increased reprogramming efficiency in induced pluripotent stem cells of mineralized tissues, but not in soft tissues. In conclusion, these results suggest that Nfic is a crucial factor for maintaining the stem cell niche of mineralized tissues and provides a possibility for Nfic as an additional factor in improving reprogramming efficiency.

Stringent regulation of the chondrocyte cell cycle is required for endochondral bone formation. During the longitudinal growth of long bones, mesenchymal stem cells condense and differentiate into chondrocytes. Epiphyseal chondrocytes sequentially differentiate to form growthplate cartilage, which is subsequently replaced with bone. Although the importance of nuclear factor 1C (Nfic) in hard tissue formation has been extensively studied, knowledge regarding its biological roles and molecular mechanisms in this process remains insufficient. Herein, we demonstrated that Nfic deficiency affects femoral growth-plate formation. Chondrocyte proliferation was downregulated and the number of apoptotic cell was increased in the growth plates of Nfic(-/-) mice. Further, the expression of the cell cycle inhibitor p21 was upregulated in the primary chondrocytes of Nfic(-/-) mice, whereas that of cyclin D1 was downregulated. Our findings suggest that Nfic may contribute to postnatal chondrocyte proliferation by inhibiting p21 expression and by increasing the stability of cyclin D1 protein.

Our previous studies indicate that NFI-C is essential for tooth root development and endochondral ossification. However, its exact role in calvarial intramembranous bone formation remains unclear. In this study, we demonstrate that the disruption of the Nfic gene leads to defects in intramembranous bone formation, characterized by decreased osteogenic proliferative activity and reduced osteoblast differentiation during postnatal osteogenesis. Additionally, Nfic-deficient mice exhibited incomplete suture closure, although Nfic disruption did not affect prenatal calvarial bone development. We found that the expression levels of Fgfr1 and Fgfr2 were reduced in the primary calvarial mesenchymal cells of Nfic-deficient mice. In contrast, NFI-C overexpression in human bone marrow stromal cells (hBMSCs) significantly increased the expression of these factors. Furthermore, NFI-C regulates FGFR1 expression by directly binding to its promoter. These results indicate that NFI-C is crucial in regulating calvarial bone formation and suture closure by controlling Fgfr1 expression and cellular proliferation.

Periodontitis is a chronic inflammatory disease characterized by persistent inflammation, redox imbalance, and mitochondrial dysfunction, leading to progressive impairment of periodontal cellular function and tissue integrity. Current periodontal therapies primarily focus on controlling inflammation and promoting tissue regeneration. However, these approaches do not adequately restore the function of resident cells compromised by an inflammatory microenvironment. Restoration of cellular homeostasis prior to inducing regeneration may enhance treatment outcomes for effective periodontal repair. This study aimed to investigate the role of CPNE7 in periodontal homeostasis and to evaluate whether selcopintide (SCPT), a CPNE7-derived peptide, restores cellular function under inflammatory periodontal conditions and exerts therapeutic effects in a mouse ligature-induced periodontitis model. In aged periodontal tissues, CPNE7 expression was reduced, accompanied by increased oxidative stress and decreased Nrf2 expression. Cpne7 knockout mice also exhibited periodontal structural abnormalities and showed aggravated alveolar bone loss and inflammatory responses following periodontitis induction. SCPT reduced excessive reactive oxygen species accumulation induced by inflammatory stimuli and restored redox balance, thereby supporting cellular homeostasis. In addition, SCPT improved mitochondrial function under inflammatory conditions, as evidenced by restored mitochondrial morphology and bioenergetic function, while attenuating inflammatory signaling and apoptosis. These protective effects were associated with activation of the Nrf2 antioxidant pathway. Importantly, topical administration of SCPT significantly attenuated periodontal destruction and alveolar bone loss in the ligature-induced periodontitis model, demonstrating its therapeutic efficacy in vivo . Collectively, these findings indicate that CPNE7 is an important contributor to periodontal tissue homeostasis and that SCPT restores redox balance, mitochondrial function, and inflammatory stability, highlighting CPNE7-derived peptides as a promising therapeutic strategy for periodontitis.