Abnormalities in esophageal smooth muscle induced by mutations in collagen XIX.

Collagen XIX is a nonfibrillar collagen that localizes in restricted tissues at very low amounts. A previous study on Col19a1 null mice revealed that collagen XIX is involved in esophageal muscle physiology and morphogenesis. Here, we use histological analysis to show that mice with a Col19a1 mutant lacking the NC3 domain and seven collagen triplets display abnormal transition of smooth to striated muscle in the abdominal segment of esophagus, and a widened esophagus with age. With two newly prepared antibodies, we analyzed the expression of collagen XIX in the mouse esophagus and show that collagen XIX colocalizes with α-smooth muscle actin. By immunoelectron microscopy, we confirmed the localization of collagen XIX in esophageal smooth muscle cells. Col19a1 mutant mice contained reduced levels of mutated Col19a1 mRNA. Interestingly, hepatocyte growth factor, which has an important role in esophageal striated muscle development, was reduced in the esophagus of the Col19a1 mutant mice. These findings suggest that collagen XIX may be critical for the function of esophageal smooth muscle cells as a scaffold for anteroposterior migration of esophagus-striated muscle cells.

MicroRNA-26 regulates the expression of CTGF after exposure to ionizing radiation.

Radiation-induced fibrosis (RIF) is a serious complication that occurs after irradiation and which is caused by the deposition of extracellular matrix (ECM) proteins such as collagen. However, the underlying mechanisms, including the expression of the cytokines, that promote the RIF process, are not yet fully understood. MicroRNAs (miRNAs) have recently been suggested to act as post-transcriptional repressors for many genes; however, their role in the process of RIF remains to be elucidated. Our previous study showed that ionizing radiation increased the type I collagen expression through the activation of transforming growth factor (TGF)-ß, while miR-29 repressed this increase. This study aimed to investigate the mechanisms by which the expression of connective tissue growth factor (CTGF), a downstream mediator of TGF-ß, is controlled by miRNAs post-transcriptionally after exposure to ionizing radiation. The expression of CTGF in NIH-3T3 cells and mouse embryonic fibroblasts was increased by ionizing radiation. However, this increase was suppressed with a specific inhibitor of TGF-ß receptor. Among the predictable miRNAs that target the CTGF gene, the expression of miR-26a was downregulated after exposure to ionizing radiation and this regulation was negatively mediated by TGF-ß signaling. miR-26a negatively regulated the CTGF expression at the post-transcriptional level; however, ionizing radiation suppressed this negative regulation. In addition, the overexpression of miR-26a inhibited the expression of CTGF and type I collagen after irradiation. In conclusion, miR-26a modulates the expression of CTGF via TGF-ß signaling in irradiated fibroblasts. The results suggest the potential application of miR-26a in the treatment of RIF.

The pro-α1(V) collagen gene (Col5a1) is coordinately regulated by miR-29b with core promoter in cultured cells.

AIMS: Col5a1 encodes the α1 chain of type V collagen, a quantitatively minor fibrillar collagen that is critical for the formation and function of the organs in the body. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate biological functions by binding to the 3'-untranslated region (3'UTR) of specific target mRNA. In this study, we investigated the posttranscriptional regulation of miRNAs on the Col5a1 gene expression. MATERIALS AND METHODS: We cultured osteoblasts and fibroblasts of cell lines. To examine the 3'UTR activity of the Col5a1 gene, chimeric plasmids constructs containing the core promoter and 3'UTR of Col5a1 were generated and luciferase assays were performed. We also evaluated the role of miRNA using constructs that were mutated at the putative binding sites of miRNA. In addition, we evaluated the endogenous mRNA and protein, and luciferase activity of the Col5a1 gene after miRNA overexpression/knockdown or CRISPR/Cas9-induced knockout. RESULTS: The luciferase assay showed a decreased activity of the 3'UTR of Col5a1 gene. However, the expression of the mutant constructs of miRNA-binding sites was restored. The overexpression of miRNA inhibited the Col5a1 gene not only with regard to the luciferase activity and endogenous mRNA but also at the protein level. In contrast, the RNAi-mediated knockdown or CRISPR/Cas9 system increased the expression of the Col5a1 gene. CONCLUSION: These results provided evidence that miR-29b regulates the Col5a1 gene expression through binding to the 3'UTR, which might play an important role in the pathogenesis of disease related to bone metabolism and fibrogenic reactions.

Regulation of type I collagen expression by microRNA-29 following ionizing radiation.

Radiation-induced fibrosis (RIF) is thought to involve the excessive accumulation of collagen and other extracellular matrix components; previously, we reported that ionizing radiation increased the type I collagen expression and that transforming growth factor (TGF)-ß was involved in this increase through activating its downstream mediator, Smad3. A recent study found that microRNAs (miRNAs)-small, noncoding sequences approximately 20 nucleotides long-negatively regulate the gene expression posttranscriptionally, and it has been suggested that miRNAs play essential roles in cellular processes, including fibrosis. However, their role in the development of RIF remains unexplored. In the present study, we examined the effects of miRNA on the expression of type I collagen induced by ionizing radiation and the mechanisms underlying the miRNA expression observed following ionizing radiation. We analyzed the regulation of miRNA following ionizing radiation by an miRNA real-time PCR, and found that miR-29 family members were downregulated in irradiated mouse fibroblasts and directly targeted type I collagen genes by specifically binding to the 3' untranslated region. We also found that the overexpression of miR-29 inhibited the ionizing radiation-induced expression of type I collagen, whereas the knockdown of miR-29 enhanced it. In addition, TGF-ß/Smad-signaling significantly decreased the transcription of miR-29, whereas the inhibition of this signaling pathway cancelled this decrease. In conclusion, miR-29 was involved in the regulation of type I collagen expression through the TGF-ß/Smad-signaling pathway in irradiated cells, suggesting that miR-29 may be an important regulator of RIF.

Effects of GABA on the expression of type I collagen gene in normal human dermal fibroblasts.

We examined the effects of GABA on type I collagen gene expression in normal human dermal fibroblasts. Real-time PCR analysis indicated GABA increased the level of type I collagen transcripts, and suppressed the expression of matrix metalloproteinase-1, which is a collagen-degrading enzyme. These results suggest GABA improves the skin elasticity by regulating type I collagen expression.

GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-ß-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells.

Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

A symptomatic Fabry disease mouse model generated by inducing globotriaosylceramide synthesis.

Fabry disease is a lysosomal storage disorder in which neutral glycosphingolipids, predominantly Gb3 (globotriaosylceramide), accumulate due to deficient α-Gal A (α-galactosidase A) activity. The GLAko (α-Gal A-knockout) mouse has been used as a model for Fabry disease, but it does not have any symptomatic abnormalities. In the present study, we generated a symptomatic mouse model (G3Stg/GLAko) by cross-breeding GLAko mice with transgenic mice expressing human Gb3 synthase. G3Stg/GLAko mice had high Gb3 levels in major organs, and their serum Gb3 level at 5-25 weeks of age was 6-10-fold higher than that in GLAko mice of the same age. G3Stg/GLAko mice showed progressive renal impairment, with albuminuria at 3 weeks of age, decreased urine osmolality at 5 weeks, polyuria at 10 weeks and increased blood urea nitrogen at 15 weeks. The urine volume and urinary albumin concentration were significantly reduced in the G3Stg/GLAko mice when human recombinant α-Gal A was administered intravenously. These data suggest that Gb3 accumulation is a primary pathogenic factor in the symptomatic phenotype of G3Stg/GLAko mice, and that this mouse line is suitable for studying the pathogenesis of Fabry disease and for preclinical studies of candidate therapies.

A new murine osteoblastic cell line immortalized with the SV40 large T antigen.

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion.

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5ß1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis.

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-ß1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-ß receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

Transient expression of mouse pro-α3(V) collagen gene (Col5a3) in wound healing.

The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.

The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-ß2 in human retinal pigment epithelial cells.

PURPOSE: Transforming growth factor (TGF)-ß is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-ß induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-ß2-induced collagen expression in human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-ß2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-ß2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-ß2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-ß2-induced Smad7 mRNA expression was evaluated. RESULTS: The biochemical blockade of PI3K/Akt activation inhibited TGF-ß2-induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-ß2 and reduced TGF-ß2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-ß2-induced Smad7 mRNA expression levels. CONCLUSIONS: The PI3K/Akt pathway plays a role in relaying the TGF-ß2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smad-dependent and Smad-independent pathways.

Long-term administration of the fungus toxin, sterigmatocystin, induces intestinal metaplasia and increases the proliferative activity of PCNA, p53, and MDM2 in the gastric mucosa of aged Mongolian gerbils.

OBJECTIVE: The causal agents of gastric cancer could include fungus toxins. Sterigmatocystin (ST), a fungus toxin, is a risk factor of gastric cancer. We investigated the effects of ST on the stomach tissues of Mongolian gerbils. METHODS: Seventy-five-week-old male Mongolian gerbils received ST ad libitum at a concentration of 0 ppb (non-treated, n = 11), 100 ppb (n = 7), or 1000 ppb (n = 13) dissolved in drinking water for a period of 24 weeks. After administration, we tested the histopathological changes and immunostaining for proliferating cell nuclear antigen (PCNA), p53, and MDM2 expression. RESULTS: We investigated the histopathological changes and determined the incidence of histopathological changes in animals with various gastric diseases after ST administration at a dose of 0 ppb (non-treated control), 100, or 1,000 ppb as follows: firstly, indices for gastritis were 18.2, 100, and 100%, those for erosion events were 9.1, 100, and 92.3%, and those for polyps were 0, 71.4, and 61.5%, respectively. These incidences in the ST-administered groups (100 or 1000 ppb) showed significant increases compared with those in the non-treated control group. And, lastly, indices for intestinal metaplasia were 0, 100, and 15.4%, respectively. Furthermore, immunostaining for PCNA, p53, and MDM2 expression showed significantly greater rates in the ST-administered groups (100 or 1000 ppb) than in the non-treated control group. CONCLUSION: The histopathological and immunohistopathological findings of this study indicate that ST exerts a marked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.

Dermatopontin promotes epidermal keratinocyte adhesion via alpha3beta1 integrin and a proteoglycan receptor.

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

Sp7/Osterix up-regulates the mouse pro-alpha3(V) collagen gene (Col5a3) during the osteoblast differentiation.

Type V collagen is a quantitatively minor collagen, but acts as critical regulator of fibril formation in the extracellular matrix. The purpose of this study is to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Sp7/Osterix is a transcription factor specifically expressed by osteoblasts and is important for osteoblast differentiation. The overexpression of Sp7/Osterix significantly increased the promoter activity and the endogenous mRNA level of the Col5a3 gene in osteoblastic cells. Conversely, a reduction of Sp7/Osterix by siRNA treatment decreased the promoter activity and the endogenous mRNA level of the Col5a3 gene. A CHIP assay confirmed that Sp7/Osterix interacted with the Col5a3 core promoter in vivo at the Sp1 binding site. The data from the experiments using the osteoblast differentiation model and the co-overexpression of Sp7/Osterix with Sp1 suggest that Sp7/Osterix promotes the expression of the collagen gene, Col5a3, and thereby playing a role in bone formation.

The pro-alpha2(XI) collagen gene is expressed in odontoblasts.

Since the dentine is analogous to bone, its extracellular matrix shares many similarities to bone tissues. Similar to the bone, type I collagen is the major organic component in dentine. However, little is known about minor fibrillar collagens, which seem to be co-expressed such as type I or II collagen. The present study examined the gene expression of type V and XI collagens, which play important roles in collagen fibril formation and skeletal morphogenesis, using RT-PCR and in situ hybridization combined with immunohistochemistry. The transcripts of pro-alpha1(XI), pro-alpha2(XI), pro-alpha1(V) and pro-alpha2(V) chains were present, but not pro-alpha3(V) and pro-alpha1(II) chains, of which an overglycosylated variant is pro-alpha3(XI) chain, in mouse incisor tooth, using RT-PCR and in situ hybridization. The pro-alpha2(XI) protein, which is mainly expressed in cartilage, were observed in the odontoblast using a specific polyclonal antibody. Real-time RT-PCR showed that the transcripts of pro-alpha2(XI), pro-alpha1(V) and pro-alpha2(V) were predominant in crown and that of pro-alpha1(XI) in root of the tooth. Finally, the expression of pro-alpha2(XI) was confirmed with an odontoblastic cell line transformed with human telomerase reverse transcriptase (hTERT) both in vitro and in vivo. The data suggest a new subtype of the V/XI collagen molecule containing alpha2(XI) chain.

The Sp1 and CBF/NF-Y transcription factors cooperatively regulate the mouse pro-alpha3(V) collagen gene (Col5a3) in osteoblastic cells.

The purpose of this study was to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Transient transfection into rat osteosarcoma ROS17/2.8 cells demonstrated that a region from nucleotides 337 to 1 was involved in the transcriptional activity of the Col5a3 gene. An electrophoretic mobility shift assay showed that Sp1/Sp3 and CBF/NF-Y bound to a GC-rich domain (194/186) and a CCAAT box (134/130) in the Col5a3 gene, respectively. Introduction of mutations or deletion into a GC-rich domain, the CCAAT box, or both elements decreased the transcription activity. Overexpression of Sp1 increases the transcription activity and interferes with Sp family binding to the GC-rich domain to decrease promoter activity. Therefore, the transcription of the mouse Col5a3 gene is cooperatively regulated by Sp1 and CBF/NF-Y in osteoblastic cells.

Preclinical efficacy and safety of 1-deoxygalactonojirimycin in mice for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of alpha-galactosidase A (alpha-Gal A) activity. It has been shown that protein misfolding is primarily responsible for the enzyme deficiency in a large proportion of mutations identified in Fabry patients with residual enzyme activity, and 1-deoxygalactonojirimycin (DGJ) can effectively increase the residual enzyme activity in cultured patient's cells. Herein, we demonstrate the preclinical efficacy and safety of DGJ in transgenic mice that express human mutant alpha-Gal A activity. alpha-Gal A activity in heart, kidney, spleen, and liver was increased dose- and time-dependently. The mutant alpha-Gal A was increased in cardiomyocytes and distal convoluted tubules of the transgenic mice in a null background after 2 weeks of DGJ treatment. Globotriaosylceramide storage was remarkably reduced in kidney of mice after a 4-week treatment at a dosage of approximately 3 mg/kg body weight/day. The half-life of DGJ was less than 1 day in all major issues and that of the enzyme synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at approximately 30 mg/kg body weight/day for 9 weeks. Furthermore, no change was observed in appearance, growth, fertility, and life span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and is well tolerated in mice.

Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX-rich basement membrane zone.

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.