Distal-less Homeobox Research Articles

Upregulation of DLX2 Confers a Poor Prognosis in Glioblastoma Patients by Inducing a Proliferative Phenotype

The human Distal-less Homeobox (DLX) gene family encodes homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis, which is primarily expressed in embryonic development. Recently, DLX gene family was reported to have essential roles in carcinogenesis. We have profiled whole genome expressed genes in 83 glioblastoma multiforme (GBM) patients from the Chinese Glioma Genome Atlas (CGGA) Group. Two major groups of samples were identified in mRNA expression profiles (referred to as Cluster 1 (C1) and Cluster 2 (C2)). We identified 7 out of the top 10 Gene Ontology terms in the C1 group were associated with differentiation and development of neuronal cell. The most significant prognostic gene was DLX2 (P < 0.001, OR = 1.744); overexpression of DLX2 indicated poor survival in the 83 GBM patients (low DLX2 vs high DLX2, 77.6 vs 44.7 weeks, P < 0.001). Annotation of mRNA profiling data on GBM from The Cancer Genome Atlas and MD Anderson Cancer Center showed the proneural and neural subtypes highly correlated with low and high DLX2 expression, respectively. Knocking down of DLX2 in GBM cell line-LN229 results in decreased cyclin D1 expression and cell proliferation. Collectively, these data identified high expression of DLX2 as a poor prognostic marker to GBM patients.

Preventing MEK1 activation influences the responses of human osteosarcoma cells to bone morphogenetic proteins 2 and 9

It was recently suggested that bone morphogenetic protein (BMP)-2 may be useful for treating osteosarcoma cells. BMP-9, which has been patented to treat breast and prostate cancers, has a higher osteoinductive potential than BMP-2. Peptides derived from the knuckle epitope of BMPs (pBMPs) also induced osteogenic differentiation. However, the effect of BMP-9 and pBMPs on osteosarcoma cells is unclear. We analyzed the effects of BMP-2, BMP-9, pBMP-2, and pBMP-9 on the behavior of human MG-63 and SaOS-2 osteosarcoma cells. An inhibitor of MEK1 activation (PD98059) that prevents downstream extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and a specific inhibitor of p38 were also used as mitogen activated protein kinase-targeting therapy is being investigated as a treatment modality for osteosarcoma. BMP-2 and BMP-9 (1.92 nmol/l) induced the phosphorylation of Smad1/5/8 in both osteosarcoma cells within 1 h but had different effects on mitogen activated protein kinase pathways. Whereas BMP-2 mainly activated ERK1/2, BMP-9 phosphorylated p38 within 1 h. pBMP-2 did not activate either the Smad or ERK/p38, whereas pBMP-9, like BMP-9, induced both Smad1/5/8 and p38 phosphorylation. p38 activation by BMP-9 or pBMP-9 was also enhanced by PD98059. However, BMP-2 or BMP-9 increased the amounts of distal-less homeobox 5 and Osterix mRNAs in SaOS-2 cells within 6 h, whereas pBMP-9 had no effect. PD98059 promoted the highest level of Osterix mRNA in SaOS-2 cells incubated with BMP-2 or BMP-9, whereas p38 inhibitor had no effect. Furthermore, PD98059 induced the lowest proliferation of MG-63 cells incubated with BMP-2, whereas p38 inhibitor did not affect the proliferation of either osteosarcoma cell line. Therefore a combination of BMP-2 or BMP-9 and an inhibitor of MEK1 may be a promising tool for regulating osteosarcoma cell behavior.

Upregulation of DLX2 Confers a Poor Prognosis in Glioblastoma Patients by Inducing a Proliferative Phenotype

The human Distal-less Homeobox (DLX) gene family encodes homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis, which is primarily expressed in embryonic development. Recently, DLX gene family was reported to have essential roles in carcinogenesis. We have profiled whole genome expressed genes in 83 glioblastoma multiforme (GBM) patients from the Chinese Glioma Genome Atlas (CGGA) Group. Two major groups of samples were identified in mRNA expression profiles (referred to as Cluster 1 (C1) and Cluster 2 (C2)). We identified 7 out of the top 10 Gene Ontology terms in the C1 group were associated with differentiation and development of neuronal cell. The most significant prognostic gene was DLX2 (P

Increased expression of DLX2 correlates with advanced stage of gastric adenocarcinoma

To investigate the expression of distal-less homeobox 2 (DLX2) in gastric adenocarcinoma and its clinicopathological significance. Gastric adenocarcinoma tissues were obtained from gastrectomy specimens of 129 patients from the Department of Surgery and Pathology, the Second Affiliated Hospital of Kunming Medical University. Sixty cases of normal gastric tissues were collected from gastrectomy specimens of adjacent gastric cancer margins greater than 5 cm. Patient diagnosis was established pathologically, and no patient had received chemotherapy or radiotherapy before surgery. All tissue specimens were formalin-fixed and paraffin-embedded. Immunohistochemistry was carried out to investigate the expression of DLX2 in 129 gastric adenocarcinoma tissues and 60 adjacent normal tissues. The immunostaining reaction was semiquantitatively evaluated based on the proportion of positive cells and the median staining intensity in normal gastric epithelial cells or tumor cells. All patients had follow-up records for more than 5 years. Correlations of DLX2 expression with clinicopathological features and prognosis of patients with gastric adenocarcinoma were analyzed. All statistical analyses were performed using the SPSS 17.0 software. The positive expression of DLX2 was detected in 68 (52.7%) cases of 129 gastric adenocarcinoma tissues and 14 (23.3%) cases of 60 adjacent normal tissues. The difference in DLX2 expression between gastric adenocarcinoma tissues and adjacent normal tissues was statistically significant (χ² = 14.391, P < 0.001). Moreover, high expression of DLX2 was detected in 48 (37.2%) cases of 129 human gastric cancer tissues, but not in adjacent normal tissues. The expression of DLX2 correlated with the size of tumor (P = 0.001), depth of invasion (P = 0.008), lymph node metastasis (P = 0.023) and tumor-node-metastasis stages (P = 0.020), but was not correlated with age, gender, histological differentiation and distant metastasis. The Kaplan-Meier survival analysis revealed that survival time of patients with high DLX2 expression was significantly shorter than that with low DLX2 expression. However, the multivariate analysis showed that invasion depth (P < 0.001), lymph nodes metastasis (P = 0.001) and distant metastasis (P < 0.001) were independent prognostic factors for patients with gastric adenocarcinoma, but DLX2 expression, tumor location and tumor size were not. These results suggest that increased expression of DLX2 may correlate with the advanced stage of gastric adenocarcinoma, and it may contribute to tumor development.

Sequence analyses of the distal-less homeobox gene family in East African cichlid fishes reveal signatures of positive selection

BackgroundGen(om)e duplication events are hypothesized as key mechanisms underlying the origin of phenotypic diversity and evolutionary innovation. The diverse and species-rich lineage of teleost fishes is a renowned example of this scenario, because of the fish-specific genome duplication. Gene families, generated by this and other gene duplication events, have been previously found to play a role in the evolution and development of innovations in cichlid fishes - a prime model system to study the genetic basis of rapid speciation, adaptation and evolutionary innovation. The distal-less homeobox genes are particularly interesting candidate genes for evolutionary novelties, such as the pharyngeal jaw apparatus and the anal fin egg-spots. Here we study the dlx repertoire in 23 East African cichlid fishes to determine the rate of evolution and the signatures of selection pressure.ResultsFour intact dlx clusters were retrieved from cichlid draft genomes. Phylogenetic analyses of these eight dlx loci in ten teleost species, followed by an in-depth analysis of 23 East African cichlid species, show that there is disparity in the rates of evolution of the dlx paralogs. Dlx3a and dlx4b are the fastest evolving dlx genes, while dlx1a and dlx6a evolved more slowly. Subsequent analyses of the nonsynonymous-synonymous substitution rate ratios indicate that dlx3b, dlx4a and dlx5a evolved under purifying selection, while signs of positive selection were found for dlx1a, dlx2a, dlx3a and dlx4b.ConclusionsOur results indicate that the dlx repertoire of teleost fishes and cichlid fishes in particular, is shaped by differential selection pressures and rates of evolution after gene duplication. Although the divergence of the dlx paralogs are putative signs of new or altered functions, comparisons with available expression patterns indicate that the three dlx loci under strong purifying selection, dlx3b, dlx4a and dlx5a, are transcribed at high levels in the cichlids’ pharyngeal jaw and anal fin. The dlx paralogs emerge as excellent candidate genes for the development of evolutionary innovations in cichlids, although further functional analyses are necessary to elucidate their respective contribution.

Distal‐less homeobox 5 inhibits adipogenic differentiation through the down‐regulation of peroxisome proliferator‐activated receptor γ expression

Distal-less homeobox 5 (Dlx5) is a positive regulator of osteoblast differentiation that contains a homeobox domain. Because there are possible reciprocal relationships between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs), we examined the regulatory role of Dlx5 in adipogenic differentiation in this study. Adipogenic stimuli suppressed the expression levels of Dlx5 mRNA in mouse bone marrow stromal cells. Over-expression of Dlx5 inhibited adipogenic differentiation in human bone marrow MSCs and 3T3-L1 preadipocytic cells whereas knockdown of Dlx5 enhanced adipogenic differentiation in 3T3-L1 cells. Over-expression of Dlx5 suppressed the expression of adipogenic marker genes, including CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Dlx5-mediated suppression of adipogenic differentiation was overcome by over-expression of PPARγ but not by that of cAMP response element binding protein (CREB) or C/EBPα. Dlx5 decreased the transcriptional activity of CREB and C/EBPα in a dose-dependent manner. Dlx5 directly bound to CREB and C/EBPα and prevented them from binding to and subsequently transactivating the PPARγ promoter. These results suggest that Dlx5 plays an important regulatory role in fate determination of bone marrow MSCs toward the osteoblast lineage through the inhibition of adipocyte differentiation as well as the direct stimulation of osteoblast differentiation.

DLX5 (distal-less homeobox 5)

Review on DLX5 (distal-less homeobox 5), with data on DNA, on the protein encoded, and where the gene is implicated.

Establishment of mesenchymal cell line derived from human developing odontoma

An odontoma, which shows proliferating odontogenic epithelium and mesenchymal tissue, is one of the most common odontogenic tumors encountered. These are commonly found in tooth-bearing regions, although the etiology remains unknown. There are no previous reports of an established line of immortalized human odontoma cells. Using odontoma fragments obtained from a girl treated at our department, we established an immortalized human odontoma cell line and investigated cell morphology, dynamic proliferation, the presence of contamination, and karyotype. Moreover, cell characterization was examined using osteogenic and odontogenic markers. We successfully established a mesenchymal odontoma cell (mOd cells). The cells were found to be fibroblastic and had a high level of telomerase activity. Cell growth was confirmed after more than 200 population doublings without significant growth retardation. mOd cells expressed mRNA for differentiation markers, including collagen type I (COLI), alkaline phosphatase, bone sialoprotein, osteopontin, osteocalcin, cementum-derived protein (CP-23), dentin sialophosphoprotein (DSPP), and distal-less homeobox 3 (DLX3), as well as bone morphogenetic proteins (BMPs). In addition, they showed a high level of calcified nodule formation activity in vitro. We successfully established a cell line that may be useful for investigating the mechanisms of normal odontogenesis as well as characteristics of odontoma tumors.

Identification of Early Response Genes to Roughness and Fluoride Modification of Titanium Implants in Human Osteoblasts

Tissue response after implantation determines the success of the healing process. This response is not only dependent on the chemical properties of the implant surface but also by the surface topography or its roughness. Although in vitro and in vivo studies show improved results with rough- and fluoride-modified implants, the mechanisms behind these findings are still unknown. Here, we have used a two-step procedure to identify novel genes related to the early response of primary human osteoblasts to roughness and fluoride-modified titanium implants. Two hundred seventeen genes responding to roughness were identified by microarray analysis and 198 genes responding to fluoride, 33 genes were common. Those identified genes related to bone and mineralization were further investigated by real-time reverse-transcriptase polymerase chain reaction. After 1 day of culture, toll-like receptor 3, ankylosis-progressive homolog, decorin, osteocalcin, and runt-related transcription factor-2 were classified as responsive genes to roughness; Distal-less homeobox-2 and Tuftelin-1 as responsive genes to fluoride treatment. Responsive genes to both treatments were collagen type I, parathyroid hormone-like hormone, hairy and enhancer of split-1, follistatin, ectonucleotide pyrophosphatase/phosphodiesterase-1, and thyroid hormone receptor-alpha. Our strategy was useful for identifying novel genes that might be involved in the early response of osteoblasts to rough and fluoride-modified titanium implants.

DLX4 (distal-less homeobox 4)

Review on DLX4 (distal-less homeobox 4), with data on DNA, on the protein encoded, and where the gene is implicated.

DLX4 Upregulates TWIST and Enhances Tumor Migration, Invasion and Metastasis

The distal-less homeobox gene 4 (DLX4) is a member of the DLX family of homeobox genes. Although absent from most normal adult tissues, DLX4 is widely expressed in leukemia, lung, breast, ovarian and prostate cancers. However the molecular targets, mechanisms and pathways that mediate the role of DLX4 in tumor metastasis are poorly understood. In this study, we found that DLX4 induces cancer cells to undergo epithelial to mesenchymal transition (EMT) through TWIST. Overexpression of DLX4 increased expression of TWIST expression in cancer cell lines, resulting in increased migratory and invasive capacity. Likewise, knocking down expression of DLX4 decreased TWIST expression and the migration ability of cancer cell lines. DLX4 bound to regulatory regions of the TWIST gene. Both western blotting and immunohistochemistry staining showed that the expression of DLX4 and TWIST are correlated in most of breast tumors. Taken together, these data from both cell models and tumor tissues demonstrate that DLX4 not only upregulates TWIST expression but also induces EMT and tumor metastasis. Altogether, we propose a new pathway in which DLX4 drives expression of TWIST to promote EMT, cancer migration, invasion and metastasis.

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Molecular and circadian controls of ameloblasts

Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities (i.e. enamel protein secretion and enamel mineralization). This time-related control is orchestrated by oscillations of clock proteins involved in the regulation of circadian rhythms. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of the transcription factors distal-less homeobox 3 (Dlx3) and runt-related transcription factor 2 (Runx2), and the clock gene nuclear receptor subfamily 1, group D, member 1 (Nr1d1), on secretory and maturation ameloblasts [using stage-specific markers amelogenin (Amelx), enamelin (Enam), and kallikrein-related peptidase 4 (Klk4)] were evaluated in the HAT-7 ameloblast cell line. Amelx and Enam steady-state mRNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 mRNA was up-regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast-specific genes but also influenced the expression of Runx2. Multiscale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.

Transcription factor Dlx2 protects from TGFβ-induced cell-cycle arrest and apoptosis

Acquiring resistance against transforming growth factor β (TGFβ)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFβ's tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFβ-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFβ receptor II (TGFβRII) gene expression and reduces canonical, Smad-dependent TGFβ signalling and expression of the cell-cycle inhibitor p21(CIP1) and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFβ from its tumour suppressive to its tumour-promoting functions.

Distal-less homeobox 3 (Dlx3) mutant mice as a model for skin inflammation

Distal-less homeobox 3 (Dlx3) mutant mice as a model for skin inflammation

Insulin-dependent diabetes mellitus decreases osteoblastogenesis associated with the inhibition of Wnt signaling through increased expression of Sost and Dkk1 and inhibition of Akt activation

Insulin-dependent diabetes mellitus (IDDM) is known to be associated with an increased risk of osteopenia. However, the cellular and molecular mechanisms for IDDM-induced alterations of the bone are not well understood. The effects of IDDM on bone metabolism were investigated using rats rendered diabetic by an injection of streptozotocin (STZ). After 4 weeks, the diabetic rats exhibited bone loss, low levels of osteocalcin, insulin-like growth factor-I (IGF-I) and bone alkaline phosphatase (ALP) activity with normal levels of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K activity, and urinary excretion of deoxypyridinoline (Dpd). Histological analysis showed a decrease in the number of osteoblasts with a normal number of osteoclasts in the metaphysis of the proximal tibia. The decreased expression of ALP, osteoclacin and collagen mRNA was associated with a decrease in the expression of runt-related transcription factor 2 (Runx2), Osterix and distal-less homeobox 5 (Dlx5) and an unaltered expression of bone morphogenic protein-2 (BMP2). The protein levels of Runx2, phosphorylated glycogen synthase kinase 3β (GSK3β), active β-catenin and β-catenin decreased. The activation of Akt was inhibited. The mRNA and protein levels of sclerosteosis (Sost) and Dickkopf 1 (Dkk1), inhibitors of Wnt signaling, increased. The mRNA expression of IGF-I and the IGF-I receptor (IGF-IR) was suppressed. These changes observed in the bone of diabetic rats were reversed by treatment with insulin, but not by normalization of the circulating IGF-I levels by treatment with IGF-I. These results suggest that insulin-deficiency in IDDM decreases osteoblastogenesis associated with inhibition of Wnt signaling through the increased expression of Sost and Dkk1 and the inhibition of Akt activation.

Neuronal activity is required for the development of specific cortical interneuron subtypes

Electrical activity has been shown to regulate development in a variety of species and in various structures, including the retina, spinal cord and cortex. Within the mammalian cortex specifically, the development of dendrites and commissural axons in pyramidal cells is activity-dependent. However, little is known about the developmental role of activity in the other major cortical population of neurons, the GABA-producing interneurons. These neurons are morphologically and functionally heterogeneous and efforts over the past decade have focused on determining the mechanisms that contribute to this diversity. It was recently discovered that 30% of all cortical interneurons arise from a relatively novel source within the ventral telencephalon, the caudal ganglionic eminence (CGE). Owing to their late birth date, these interneurons populate the cortex only after the majority of other interneurons and pyramidal cells are already in place and have started to functionally integrate. Here we demonstrate in mice that for CGE-derived reelin (Re)-positive and calretinin (Cr)-positive (but not vasoactive intestinal peptide (VIP)-positive) interneurons, activity is essential before postnatal day 3 for correct migration, and that after postnatal day 3, glutamate-mediated activity controls the development of their axons and dendrites. Furthermore, we show that the engulfment and cell motility 1 gene (Elmo1), a target of the transcription factor distal-less homeobox 1 (Dlx1), is selectively expressed in Re(+) and Cr(+) interneurons and is both necessary and sufficient for activity-dependent interneuron migration. Our findings reveal a selective requirement for activity in shaping the cortical integration of specific neuronal subtypes.

Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7-21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.

Ossification process involving the human thoracic ligamentum flavum: role of transcription factors

IntroductionOssification of the ligamentum flavum (OLF) of the spine is associated with serious neurologic compromise, but the pathomechanism of this process remains unclear. The objective of this study was to investigate the pathomechanism of the ossification process, including the roles of various transcriptional factors in the ossification of human thoracic ligamentum flavum.MethodsSections of the thoracic ligamentum flavum were obtained from 31 patients with OLF who underwent posterior thoracic decompression, and from six control patients free of OLF. Cultured ligamentum flavum cells (n = 6, each) were examined with real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis for Sry-type high-mobility group box 9 (Sox9), runt-related transcription factor 2 (Runx2), muscle segment homeobox 2 (Msx2), Osterix, distal-less homeobox 5 (Dlx5), and AP-1. The harvested sections were examined with hematoxylin-eosin, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, and immunohistochemistry for the transcriptional factors.ResultsCompared with the control, the OLF showed disorganization of the elastic fiber bundles and abundant hypertrophic chondrocytes in the ossification front. TUNEL-positive chondrocytes were found near the ossified plaques. The mRNA expression levels of Sox9, Runx2, Msx2, and AP-1 in cultured cells from the ligamentum flavum of OLF patients were significantly different from those of the control. OLF samples were strongly immunoreactive to Sox9, Runx2, and Msx2 at proliferating chondrocytes in the fibrocartilage area. Hypertrophic chondrocytes were positive for Runx2, Osterix, Dlx5, and AP-1.ConclusionsThe ossification process in OLF seems to involve chondrocyte differentiation under the unique expression of transcriptional factors. Accumulation of hypertrophic chondrocytes was evident around the calcified area at the ossification front, and we suggest that the differentiation of these cells seems to be concerned with the ossification process.

Tumor Necrosis Factor-α Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-Dlx5 Pathway

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × 10(4)) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.