A low-calcium microenvironment is imperative for spermatozoa maturation within the epididymis. Our previous work has shown that γ-glutamyl carboxylase (GGCX), the carboxylation enzyme of the matrix Gla protein (MGP), plays an essential role in epididymal calcium homeostasis and sperm maturation in rats and that the GGCX SNP mutation rs699664 was associated with asthenozoospermia (AZS) in humans. Here, we investigated the expression patterns of GGCX and MGP in the mouse epididymis and generated GgcxK325Q knock-in (KI) mice. We also tested the effects of this mutation on epididymal calcium homeostasis, sperm function, and male fertility in GgcxK325Q-/- mice. The results showed that both GGCX and MGP were enriched in all regions of the mouse epididymis, especially in the initial segment of the epididymis. Double immunofluorescence staining revealed that GGCX colocalized with MGP in the epithelial cells of the initial segment and caput regions as well as in the lumen of the corpus and cauda regions of the mouse epididymis. However, the GgcxK325Q-/- mice were fertile with normal epididymal morphology, sperm functions, and epididymal calcium concentration. Overall, our findings revealed that the GgcxK325Q mutation does not exert any discernible effect on male fertility in mice.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common craniofacial birth defect with complex etiologies. Recently, the dysregulation of long noncoding RNAs (lncRNAs) has been implicated in many developmental diseases, including NSCL/P. However, the functions and mechanisms of lncRNAs in NSCL/P have not been fully elucidated. In this study, we found that lncRNA MIR31HG in NSCL/P patients was significantly downregulated than that in healthy individuals (GSE42589, GSE183527). In addition, single nucleotide polymorphism rs58751040 in MIR31HG was nominally associated with NSCL/P susceptibility (odds ratio: 1.29, 95% confidence interval: 1.03-1.54, p = 4.93 × 10-2) through a case-control study (504 NSCL/P cases and 455 controls). Luciferase activity assay showed that the C allele of rs58751040 revealed a decreased transcription activity of MIR31HG than the G allele. Moreover, knockdown of MIR31HG promoted cell proliferation and migration in human oral keratinocytes and human embryonic palate mesenchyme. Bioinformatic analysis and cellular studies suggested that MIR31HG may confer susceptibility to risk of NSCL/P through matrix Gla protein (MGP) signaling. In summary, we identified a novel lncRNA involved in the development of NSCL/P.

UNASSIGNED: The contribution of inactive Matrix Gla protein (MGP) to ectopic vascular calcification associated with type 2 diabetes mellitus (T2DM) is well recognized. However, its role in diabetic microvascular complications remains unknown. The study aim was to identify any association between inactive MGP and diabetic retinopathy (DR). Its relation to insulin resistance was also explored.
UNASSIGNED: The study included 90 participants, 65 Type 2 diabetic patients (25 without DR and 40 with DR) and 25 healthy controls. Serum inactive MGP was measured using ELISA. HOMA-IR was also assessed.
UNASSIGNED: Inactive MGP was significantly higher in both diabetic groups compared to controls (P < 0.001), as well as in Type 2 diabetic patients with retinopathy compared to Type 2 diabetes without retinopathy (P = 0.002). Inactive MGP was positively correlated with HbA1c, HOMA-IR, LDL-C and triglycerides (P < 0.001), and negatively correlated with HDL-C (P = 0.008) and eGFR (P < 0.001). Logistic Regression Analysis showed that inactive MGP was one of the most associated factors with DR.
UNASSIGNED: Inactive MGP was found to be related to DR, insulin resistance and other dysmetabolic risk factors. These findings highlight that inactive MGP may be a significant contributor to the pathogenesis, evolution, and progression of DR.

An imbalance of human mesenchymal stem cells (MSCs) adipogenic and osteogenic differentiation plays an important role in the pathogenesis of osteoporosis. Our previous study verified that Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1)/myoferlin deficiency promotes adipogenic differentiation of MSCs by blocking autophagic flux in osteoporosis. However, the function of APPL1 in the osteogenic differentiation of MSCs remains unclear. This study aimed to investigate the role of APPL1 in the osteogenic differentiation of MSCs in osteoporosis and the underlying regulatory mechanism. In this study, we demonstrated the downregulation of APPL1 expression in patients with osteoporosis and osteoporosis mice. The severity of clinical osteoporosis was negatively correlated with the expression of APPL1 in bone marrow MSCs. We found that APPL1 positively regulates the osteogenic differentiation of MSCs in vitro and in vivo. Moreover, RNA sequencing showed that the expression of MGP, an osteocalcin/matrix Gla family member, was significantly upregulated after APPL1 knockdown. Mechanistically, our study showed that reduced APPL1 impaired the osteogenic differentiation of mesenchymal stem cells by facilitating Matrix Gla protein expression to disrupt the BMP2 pathway in osteoporosis. We also evaluated the significance of APPL1 in promoting osteogenesis in a mouse model of osteoporosis. These results suggest that APPL1 may be an important target for the diagnosis and treatment of osteoporosis.

Metastatic breast carcinoma is commonly considered during differential diagnosis when metastatic disease is detected in females. In addition to the tumor morphology and documented clinical history, sensitive and specific immunohistochemical (IHC) markers such as GCDFP-15, mammaglobin, and GATA3 are helpful for determining breast origin. However, these markers are reported to show lower sensitivity in certain subtypes, such as triple-negative breast cancer (TNBC).
Using bioinformatics analyses, we identified a potential diagnostic panel to determine breast origin: matrix Gla protein (MGP), transcriptional repressor GATA binding 1 (TRPS1), and GATA-binding protein 3 (GATA3). We compared MGP, TRPS1, and GATA3 expression in different subtypes of breast carcinoma of (n = 1201) using IHC. As a newly identified marker, MGP expression was also evaluated in solid tumors (n = 2384) and normal tissues (n = 1351) from different organs.
MGP and TRPS1 had comparable positive expression in HER2-positive (91.2% vs. 92.0%, p = 0.79) and TNBC subtypes (87.3% vs. 91.2%, p = 0.18). GATA3 expression was lower than MGP (p < 0.001) or TRPS1 (p < 0.001), especially in HER2-positive (77.0%, p < 0.001) and TNBC (43.3%, p < 0.001) subtypes. TRPS1 had the highest positivity rate (97.9%) in metaplastic TNBCs, followed by MGP (88.6%), while only 47.1% of metaplastic TNBCs were positive for GATA3. When using MGP, GATA3, and TRPS1 as a novel IHC panel, 93.0% of breast carcinomas were positive for at least two markers, and only 9 cases were negative for all three markers. MGP was detected in 36 cases (3.0%) that were negative for both GATA3 and TRPS1. MGP showed mild-to-moderate positive expression in normal hepatocytes, renal tubules, as well as 31.1% (99/318) of hepatocellular carcinomas. Rare cases (0.6-5%) had focal MGP expression in renal, ovarian, lung, urothelial, and cholangiocarcinomas.
Our findings suggest that MGP is a newly identified sensitive IHC marker to support breast origin. MGP, TRPS1, and GATA3 could be applied as a reliable diagnostic panel to determine breast origin in clinical practice.

Matrix Gla protein (MGP) was originally reported as a physiological suppressor of ectopia calcification and has also been reported to be associated with cancer. However, the relation between the biological functions of MGP and the immune response in colorectal cancer (CRC) remains unclear. Here, we investigated the regulatory role of MGP in the immune microenvironment of CRC. MGP expression in CRC samples was assessed by single-cell RNA sequencing and the Gene Expression Omnibus (GEO) database, and confirmed by quantitative real-time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry analysis of human CRC samples. The effect of MGP on proliferation and invasion of CRC cells was evaluated by in vitro assays involving MGP knockdown and overexpression. Luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay were performed to identify transcriptional regulatory sites of the nuclear factor kappa-B (NF-κB) and programmed cell death ligand 1 (PD-L1). In vivo experiments were performed in mouse model of CRC liver metastasis established via spleen injection. The results revealed that MGP was significantly upregulated in cancer cell clusters from the primary CRC or liver metastases, compared with that in the corresponding paracancerous tissues via single-cell RNA sequencing. MGP enriched intracellular free Ca2+ levels and promoted NF-κB phosphorylation, thereby activated PD-L1 expression to promote CD8+ T cell exhaustion in CRC. The luciferase reporter assay and ChIP-qPCR assay indicated that the transcriptional regulation of NF-κB upregulated PD-L1 expression. In vivo, MGP inhibition significantly decreased the rate of CRC liver metastasis, which was further reduced after combined therapy with αPD1 (anti-PD1). In conclusions, this study revealed that MGP can facilitate CD8+ T cell exhaustion by activating the NF-κB pathway, leading to liver metastasis of CRC. The combination of MGP knockdown and αPD1 can synergistically resist liver metastasis of CRC.

Perivascular adipose-derived stem cells (PV-ADSCs) could differentiate into smooth muscle cells (SMCs), participating in vascular remodeling. However, its underlying mechanism is not well explored. Our previous single-cell RNA-sequencing dataset identified a unique expression of matrix Gla protein (MGP) in PV-ADSCs compared with subcutaneous ADSCs. MGP involves in regulating SMC behaviors in vascular calcification and atherosclerosis. In this study, we investigated MGP\'s role in PV-ADSCs differentiation toward SMCs in vitro and in vascular remodeling in vivo. PV-ADSCs were isolated from perivascular regions of mouse aortas. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence confirmed higher MGP expression in PV-ADSCs. The MGP secretion increased along PV-ADSCs differentiation toward SMCs in response to transforming growth factor-beta 1 (TGF-β1). Lentivirus knockdown of MGP markedly promoted the bone morphogenetic protein 2 (BMP2) expression and phosphorylation of SMAD1/5/8 in PV-ADSCs, subsequently inhibiting its differentiation toward SMCs. Such inhibition could be partially reversed by further application of BMP2 inhibitors. On the contrary, exogenous MGP inhibited BMP2 expression and SMAD1/5/8 phosphorylation in PV-ADSCs, thereby promoting its differentiation toward SMCs. Transplantation of cultured PV-ADSCs, which was pretreated by MGP knockdown, in mouse femoral artery guide-wire injury model significantly alleviated neointimal hyperplasia. In conclusion, MGP promoted the differentiation of PV-ADSCs toward SMCs through BMP2/SMAD-mediated signaling pathway. This study offers a supplement to the society of perivascular tissues and PV-ADSCs.

Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX) gene. The GGCX enzyme catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins, which have function in blood coagulation, calcification, and cell signaling. Therefore, in addition to bleedings, some VKCFD1 patients develop diverse non-hemorrhagic phenotypes such as skin hyper-laxity, skeletal dysmorphologies, and/or cardiac defects. Recent studies showed that GGCX mutations differentially effect γ-carboxylation of VKD proteins, where clotting factors are sufficiently γ-carboxylated, but not certain non-hemostatic VKD proteins. This could be one reason for the development of diverse phenotypes. The major manifestation of non-hemorrhagic phenotypes in VKCFD1 patients are mineralization defects. Therefore, the mechanism of regulation of calcification by specific VKD proteins as matrix Gla protein (MGP) and Gla-rich protein (GRP) in physiological and pathological conditions is of high interest. This will also help to understand the patho-mechanism of VKCFD1 phenotypes and to deduce new treatment strategies. In the present review article, we have summarized the recent findings on the function of GRP and MGP and how these proteins influence the development of non-hemorrhagic phenotypes in VKCFD1 patients.

BACKGROUND: Vitamin K-dependent proteins (VKDPs) and the epidermal growth factor receptor (EGFR) are involved in lung cancer progression. Therefore, we aimed to study the serum concentration of Matrix Gla protein (MGP), Growth Arrest-specific 6 (Gas6), and EGFR before and after the first cycle of chemotherapy and to investigate how MGP, Gas6, and EGFR are modified after one cycle of chemotherapy.
METHODS: We performed an observational study on twenty patients diagnosed with lung cancer, by assessing the serum concentration of vitaminK1 (VitK1), MGP, Gas6, and EGFR using the ELISA technique before and after three weeks of the first cycle of chemotherapy. Patients were evaluated using RECIST 1.1 criteria.
RESULTS: Serum levels of MGP, Gas6, EGFR, and VK1 before and after treatment were not changed significantly. Regarding the pre-treatment correlation of the MGP values, we found a strong positive relationship between MGP and VK1 pre-treatment values (r = 0.821, 95%CI 0.523; 0.954, p < 0.001). Furthermore, there was a moderately negative correlation between VK1 and EGFR pre-treatment values, with the relationship between them being marginally significant (r = -0.430, 95%CI -0.772; 0.001, p = 0.058). Post-treatment, we found a strong positive relationship between MGP and VK1 post-treatment values (r = 0.758, 95%CI 0.436; 0.900, p < 0.001). We also found a moderate positive relationship between Gas6 and EGFR post-treatment values, but the correlation was only marginally significant (r = 0.442, p = 0.051).

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.