Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Differential expression of basement membrane type IV collagen alpha chains in gastric intramucosal neoplastic lesions.

BACKGROUND: The histological diagnosis of gastric intramucosal neoplastic lesions (GINLs) is controversial among experienced pathologists. Although the destruction of the epithelial basement membrane (BM) and the invasion of neoplastic epithelial cells into the interstitium of the lamina propria is distinct proof of "intramucosal carcinoma," histological evaluation of GINLs is difficult and ambiguous, especially intestinal-type adenocarcinoma. Type IV collagen is a major component of the BM, and comprises six genetically distinct alpha(IV) chains, alpha1(IV) to alpha6(IV). In several types of carcinomas, the loss of alpha5/alpha6(IV) chains of the epithelial BM at an early invasive stage has been reported. However, the expression of alpha5/alpha6(IV) chains in GINLs is still unclear. We examined the immunohistochemical expression of alpha(IV) chains in GINLs and investigated whether the expression pattern was a diagnostic marker of gastric intramucosal carcinoma. METHODS: The expression of alpha(IV) chains and Ki-67 in 60 resected GINL specimens was immunohistochemically examined. RESULTS: In normal gastric epithelium, alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains were expressed continuously in the BM. In most tubular adenomas (Japanese classification), these four chains were stained continuously in the BM, whereas in tubular adenocarcinomas (Japanese classification), the alpha5/alpha6(IV) chains had disappeared, partially or completely. The expression of alpha5/alpha6(IV) chains was closely related to the grade of histological atypia. In addition, the loss of alpha5/alpha6(IV) chains was significantly correlated with tumor cell growth activity. CONCLUSIONS: The loss of alpha5/alpha6(IV) chains might be a useful diagnostic finding for gastric intramucosal carcinoma in GINL cases.

Differential expression of basement membrane collagen-IV alpha1 to alpha6 chains during oral carcinogenesis.

This study aimed to resolve if basement membrane (BM) collagen alpha chains undergo remodeling during oral carcinogenesis. Using immunohistochemistry and transmission electron microscopy, we found that BMs in oral epithelial dysplasias (OED: mild, n=10; moderate, n=10; severe, n=10) and carcinoma in situ (CIS) (n=10) differed from normal mucosa (n=6) and oral epithelial hyperplasia (n=5) in showing: (1) excessive lamina densa-like material ultrastructurally, and (2) stronger immunoexpression for alpha5(IV) than for alpha1(IV), alpha2(IV), and alpha6(IV) chains-findings that implicate these molecules' role as an adhesive template for the attachment and persistence of basal dysplastic cells. Incipient loss of BM integrity in CIS, where alpha5(IV)/alpha6(IV) chains were more frequently absent than alpha1(IV)/alpha2(IV) chains, suggests that alpha(IV) network disruption is crucial for progression of dysplastic cells into the extracellular compartment, marking transition into the invasive phase. In carcinomatous BM, the disappearance of alpha(IV) chains was more severe in poorly differentiated oral squamous cell carcinoma (OSCC) (n=10) than in well-differentiated OSCC (n=10). In all samples examined, alpha3(IV) and alpha4(IV) chains were absent. These findings taken together suggest that BM collagen-IV alpha chains undergo remodeling where selective increase and loss of these molecules are probably early and late events, respectively, during progression of oral dysplasia to cancer.

Differential expression of basement membrane type-IV collagen alpha1, alpha2, alpha5 and alpha6 chains among the histological subtypes of adenoid cystic carcinoma.

Six distinct alpha(IV) chains in the basement membrane (BM) of adenoid cystic carcinoma (ACC) of the salivary gland were immunohistochemically examined by anti-alpha(IV) chain-specific antibodies, and their expressions were compared with the histological subtypes and the expressions of cytokeratin 19 (CK19), cytokeratin 14 (CK14) and alpha-smooth muscle actin (alpha-SMA) and Ki-67. In the BM of normal salivary ducts, alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains were continuously stained, but alpha3(IV) and alpha4(IV) chains were negative. In the tubular and cribriform subtypes of ACC, tubules with continuous staining of alpha5(IV) and alpha6(IV) chains showed the biphasic-staining pattern among the expressions of CK19, CK14 and alpha-SMA. However, in cancer-cell nests with discontinuous or negative staining of alpha5(IV) and alpha6(IV) chains, the biphasic pattern was ambiguous. In the solid subtype, the staining of alpha1(IV) and alpha2(IV) chains was discontinuous, the staining of alpha5(IV) and alpha6(IV) chains was negative and the biphasic-staining pattern was unclear. The mitotic activity of cancer cells analyzed by the Ki-67 labeling index was significantly related to the expression of alpha5(IV) and alpha6(IV) chains in the cribriform subtype. These results suggest that BM irregularity with the differential expression of alpha(IV) chains in ACC closely relates to cell proliferation, cell differentiation and histological structure.

Non-helical type IV collagen polypeptides in human placenta.

Our previous reports showed that cultured human cells secrete non-disulfide-bonded non-helical alpha1(IV) and alpha2(IV) chains under physiological conditions. In the present report we show that the alpha(IV) chains in non-helical form were reactive to lectin ABA (Agaricus bisporus agglutinin), whereas the alpha(IV) chains secreted in triple-helical form were not. These results indicate that ABA could be used to distinguish the two conformational isomers of type IV collagen polypeptides. An alpha1(IV) chain isolated from human placenta with an antibody-coupled column showed a positive reaction to ABA, indicating that gelatin form of the type IV collagen alpha1(IV) chain is produced and retained in the tissue in vivo. A possible significance of the gelatin form is discussed from the finding that the non-helical alpha1(IV) chain purified with EDTA-free buffer contained degraded polypeptides including NC1-size domain and showed an apparent inhibition against activated pro-MMP-9. This is the first report to show that a gelatin form of protein exists in vivo.

Tissue- and developmental stage-specific activation of alpha 5 and alpha 6(IV) collagen expression in the upper gastrointestinal tract of transgenic mice.

Little is known about mechanisms regulating gene expression for the alpha chains of basement membrane type IV collagen, arranged head-to-head in transcription units COL4A1-COL4A2, COL4A3-COL4A4, and COL4A5-COL4A6, and implicated broadly in genetic diseases. To investigate these mechanisms, we generated transgenic mouse lines bearing 5'-flanking sequences of COL4A5 and COL4A6, cloned upstream of a lacZ reporter gene. A 3.8-kb fragment upstream of COL4A6 directs reporter gene expression in the esophagus, stomach, and duodenum, whereas a 13.8-kb fragment directs expression in the esophagus only. A 10.6-kb fragment upstream of COL4A5 directs expression in the esophagus. Coupled with evidence of long-range conservation between human and mouse non-coding sequences, described herein, our findings provide the first indication that highly specialized patterns characteristic of COL4A5-COL4A6 expression in vivo arise from effects of distributed cis-acting regulatory elements on a bidirectional proximal promoter, itself transcriptionally competent.

Differential expression of collagen IV alpha1 to alpha6 chains in basement membranes of benign and malignant odontogenic tumors.

Type IV collagen, the major component of basement membrane (BM), demonstrates a stage- and position-specific distribution of its isoforms during tooth development. To determine its localization in BM of odontogenic neoplasms, immunohistochemistry using six anti-alpha(IV) chain-specific monoclonal antibodies was performed. Results disclosed that BM demonstrated an irregular alpha(IV) chain profile in malignant odontogenic tumors as compared to benign odontogenic neoplasms. No alpha3(IV) chains were detected. Expression of alpha1(IV)/alpha2(IV) and alpha5(IV)/alpha6(IV) chains was stronger in desmoplastic ( n=3) than in ordinary (n=5) ameloblastomas. The adenomatoid odontogenic tumor ( n=2) distinctly expressed these chains in BM of cribriform areas and hyaline materials (which was also alpha4(IV)-positive), but weakly around epithelial whorls/rosettes/nests and mineralized foci. These five chains also stained BM and tumor cells of ameloblastic fibroma ( n=3) and ameloblastic fibro-odontosarcoma (n=1), but not the inductive hard tissues. Ameloblastic carcinoma ( n=2) showed specific alpha1(IV)/alpha2(IV) chain loss, while primary intraosseous carcinoma ( n=1) demonstrated a discontinuous alpha1(IV)/alpha2(IV) and alpha5(IV)/alpha6(IV) staining pattern. The present results suggest that modification and remodeling of BM collagen IValpha chains occur during odontogenic neoplasms' progression.

Distribution of basement membrane type IV collagen alpha chains in ameloblastoma: an immunofluorescence study.

BACKGROUND: Type IV collagen, a heterotrimeric molecule that exists in six genetically distinct forms, alpha1(IV)-alpha6(IV) is a major structural component of basement membrane (BM) and acts as a scaffold for other BM constituents. METHODS: Indirect immunofluorescence using alpha chain-specific monoclonal antibodies was employed to clarify basement membrane (BM) collagen IV distribution in two ameloblastoma, and for comparison, on oral mucosa and tooth germ. RESULTS: Ameloblastoma BM expressed five of six genetically distinct forms of collagen IV: alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains occurred as intense linear stainings without disruption around neoplastic epithelium, and this expression pattern was fundamentally similar to oral mucosa BM; alpha4(IV) expression was rare and occurred around nests of primitive tumor cells or potentially invasive sites. The tooth germ demonstrated a stage- and position-specific collagen IV distribution: the inner enamel epithelium BM expressed alpha1(IV), alpha2(IV), and alpha4(IV) except in the cuspal predentine region; and the outer enamel epithelium BM expressed alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains. CONCLUSIONS: Results suggest that collagen IV alpha chain distribution in ameloblastoma BM plays an important role in tumor cytodifferentiation and progression.

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors.

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.