Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

STEERing an IDeA in Undergraduate Research at a Rural Research Intensive University.

This study documents outcomes, including student career choices, of the North Dakota Institutional Development Award Networks of Biomedical Research Excellence program that provides 10-week, summer undergraduate research experiences at the University of North Dakota School of Medicine and Health Sciences. Program evaluation initiated in 2008 and, to date, 335 students have completed the program. Of the 335, 214 students have successfully completed their bachelor's degree, 102 are still undergraduates, and 19 either did not complete a bachelor's degree or were lost to follow-up. The program was able to track 200 of the 214 students for education and career choices following graduation. Of these 200, 76% continued in postgraduate health-related education; 34.0% and 20.5% are enrolled in or have completed MD or PhD programs, respectively. Other postbaccalaureate pursuits included careers in pharmacy, optometry, dentistry, public health, physical therapy, nurse practitioner, and physician's assistant, accounting for an additional 21.5%. Most students electing to stop formal education at the bachelor's degree also entered fields related to health care or science, technology, engineering, and mathematics (19.5%), with only a small number of the 200 students tracked going into service or industries which lacked an association with the health-care workforce (4.5%). These student outcomes support the concept that participation in summer undergraduate research boosts efforts to populate the pipeline of future researchers and health professionals. It is also an indication that future researchers and health professionals will be able to communicate the value of research in their professional and social associations. The report also discusses best practices and issues in summer undergraduate research for students originating from rural environments.

Elevated connexin 43 expression in arsenite-and cadmium-transformed human bladder cancer cells, tumor transplants and selected high grade human bladder cancers.

Connexin 43 has been shown to play a role in cell migration and invasion; however, its role in bladder cancer is not well defined. Previous studies from our laboratory have shown that the environmental pollutants arsenite and cadmium can cause malignant transformation of the immortalized urothelial cell line UROtsa. These transformed cells can form tumors in immune-compromised mice. The goal of the present study was to determine if connexin 43 is expressed in the normal human bladder, the arsenite and cadmiun-transformed UROtsa cells as well as human urothelial cancer. The results obtained showed that connexin 43 is not expressed in the epithelial cells of the human bladder but is expressed in immortalized cultures of human urothelial cells and the expression is variable in the arsenite and cadmium- transformed urothelial cell lines derived from these immortalized cells. Tumor heterotransplants generated from the transformed cells expressed connexin 43 and the expression was localized to areas of squamous differentiation. Immuno-histochemical analysis of human bladder cancers also showed that the expression of connexin 43 was localized to areas of the tumor that showed early features of squamous differentiation. Treatment of UROtsa cells with various concentrations of arsenite or cadmium did not significantly alter the expression level of connexin 43. In conclusion, our results show that the expression of connexin 43 is localized to the areas of the tumor that show squamous differentiation, which may be an indicator of poor prognosis. This suggests that connexin 43 has the potential to be developed as a biomarker for bladder cancer that may have the ability to invade and metastasize.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

BACKGROUND: Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. METHODS: Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. RESULTS: This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. CONCLUSIONS: The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells "seeding" a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

BACKGROUND: This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. METHODS: Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. RESULTS: It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. CONCLUSIONS: Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

Increased neuron specific enolase expression by urothelial cells exposed to or malignantly transformed by exposure to Cd²âº or As³âº.

Neuron specific enolase (ENO2, γ-enolase) is a biomarker used to help identify neuroendocrine differentiation in tumors. This laboratory has shown that ENO2 might be a biomarker for exposure to cadmium and arsenite. In this study these observations are extended to the urothelial cell, where environmental exposures are strongly linked to urothelial cancer. The UROtsa urothelial cell line and its Cd²âº- and As³âº-transformed counterparts were used as the model. Acute exposure of the UROtsa cells to both As³âº- and Cd²âº-caused significant increases in ENO2 expression. Treatment with the histone deacetlyase inhibitor was also shown to significantly increase the expression of ENO2 mRNA. The expression of ENO2 was significantly elevated in the Cd²âº- and As³âº-transformed UROtsa cells and tumor transplants. In contrast, ENO1, was unaffected by exposure to As³âº or Cd²âº. Immunofluorescence showed ENO2 associated with both the nucleus and cytoplasm and cytoplasmic ENO2 co-localized with ENO1. The findings extend the evidence suggesting a link between As³âº and Cd²âº exposure and neuroendocrine differentiation in tumors. The results suggest that ENO2 might be a biomarker of human exposure to Cd²âº and As³âº that operates through histone modification.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer.

BACKGROUND: ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. RESULTS: It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. CONCLUSION: This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease.

SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling.

Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer.

BACKGROUND: Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As+3 and Cd+2 sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants. RESULTS: It was shown that both As+3 and Cd+2 exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As+3 or Cd+2. Localization studies showed that ENO2 in the MCF-10A cells transformed by As+3 or Cd+2 had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1. CONCLUSION: The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As+3 or Cd+2. The findings also suggest a possible link between As+3 and Cd+2 exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As+3 and Cd+2.

Comparison of expression patterns of keratin 6, 7, 16, 17, and 19 within multiple independent isolates of As(+3)- and Cd (+2)-induced bladder cancer : keratin 6, 7, 16, 17, and 19 in bladder cancer.

This laboratory has generated a series of seven cadmium (Cd(+2))- and six arsenite (As(+3))-transformed urothelial cancer cell lines by exposure of parental UROtsa cells to each agent under similar conditions of exposure. In this study, the seven Cd(+2)-transformed cell lines were characterized for the expression of keratin 6, 16, and 17 while the six As(+3) cell lines were assessed for the expression of keratin 7 and 19. The results showed that the series of Cd(+2)-transformed cell lines and their respective transplants all had expression of keratin 6, 16, and 17 mRNA and protein. The expression of keratin 6, 16, and 17 was also correlated with areas of the urothelial tumor cells that had undergone squamous differentiation. The results also showed that four of the six As(+3)-transformed cell lines had expression of keratin 7 and 19 mRNA and protein and produced subcutaneous tumors with intense focal staining for keratin 7 and 19. The other two As(+3)-transformed cell lines had very low expression of keratin 7 mRNA and protein and produced subcutaneous tumors having no immunoreactivity for keratin 7; although keratin 19 expression was still present. The peritoneal tumors produced by one of these two cell lines regained expression of keratin 7 protein. The present results, coupled with previous studies, indicate that malignant transformation of UROtsa cells by Cd(+2) or As(+3) produce similar patterns of keratin 6, 7, 16, 17, and 19 in the resulting series of cell lines and their respective tumors.

SPARC gene expression is repressed in human urothelial cells (UROtsa) exposed to or malignantly transformed by cadmium or arsenite.

SPARC belongs to a class of extracellular matrix-associated proteins that have counteradhesive properties. The ability of SPARC to modulate cell-cell and cell-matrix interactions provides a strong rationale for studies designed to determine its expression in cancer. The objective of this study was to determine if SPARC expression was altered in cadmium (Cd(2+)) and arsenite (As(3+)) induced bladder cancer and if these alterations were present in archival specimens of human bladder cancer. The expression of SPARC was determined in human parental UROtsa cells, their Cd(2+) and As(3+) transformed counterparts and derived tumors, and in archival specimens of human bladder cancer using a combination of real time reverse transcriptase polymerase chain reaction, Western blotting, immunofluorescence localization and immunohistochemical staining. It was demonstrated that SPARC expression was down-regulated in Cd(2+) and As(3+) transformed UROtsa cells. In addition, the malignant epithelial component of tumors derived from these cell lines were also down-regulated for SPARC expression, but the stromal cells recruited to these tumors was highly reactive for SPARC. This finding was shown to translate to specimens of human bladder cancer where tumor cells were SPARC negative, but stromal cells were positive. Acute exposure of UROtsa cells to both cadmium and arsenite reduced the expression of SPARC through a mechanism that did not involve changes in DNA methylation or histone acetylation. These studies suggest that environmental exposure to As(3+) or Cd(2+) can alter cell-cell and cell-matrix interactions in normal urothelial cells through a reduction in the expression of SPARC. The SPARC associated loss of cell-cell and cell-matrix contacts may participate in the multi-step process of bladder carcinogenesis.

Beclin-1 expression in normal bladder and in Cd2+ and As3+ exposed and transformed human urothelial cells (UROtsa).

The expression of beclin-1 in normal human bladder and in Cd(2+) and As(3+) exposed and transformed urothelial cells (UROtsa) was examined in this study. It was shown using a combination of real-time PCR, Western analysis and immunohistochemistry that beclin-1 was expressed in the urothelial cells of the normal bladder. It was also demonstrated that the parental UROtsa cell line expressed beclin-1 mRNA and protein at levels similar to that of the in situ urothelium. The level of beclin-1 expression underwent only modest alterations when the UROtsa cells were malignantly transformed by Cd(2+) or As(3+) or when the parental cells were exposed acutely to Cd(2+) or As(3+). While there were instances of significant alterations at individual time points and within cell line-to-cell line comparisons there was no evidence of a dose-response relationship or correlations to the phenotypic properties of the cell lines. Similar results were obtained for the expression of the Atg-5, Atg-7, Atg-12 and LC3B autophagy-related proteins. The findings provide initial evidence for beclin-1 expression in normal bladder and that large alterations in the expression of beclin-1 and associated proteins do not occur when human urothelial cells are malignantly transformed with, or exposed to, either Cd(2+) or As(3+.).

Keratin 6 expression correlates to areas of squamous differentiation in multiple independent isolates of As(+3)-induced bladder cancer.

This laboratory has shown that arsenite (As(+3)) exposure can cause the malignant transformation of the UROtsa human urothelial cell line. This single isolate formed subcutaneous tumors with a histology similar to human urothelial cell carcinoma. The tumors also displayed areas of squamous differentiation of the urothelial cells, an infrequent but known component of human bladder cancer. In the present study, five additional independent isolates of As(+3)-transformed urothelial cells were isolated and each was shown to produce subcutaneous urothelial cell tumors with a characteristic histology very similar to those described in the initial report. That there were underlying phenotypic differences in the six independent isolates was demonstrated when they were assessed for their ability to form tumors within the peritoneal cavity. It was shown that two isolates could form hundreds of small peritoneal tumor nodules, one isolate a moderate number of tumor nodules, and three isolates no or only one tumor nodule. The peritoneal tumors were also characterized for their degree of squamous differentiation of the urothelial cells and, while areas of squamous differentiation could be found, such differentiation was substantially reduced compared to subcutaneous tumors. Immunostaining for keratin 6 was tested as a potential marker for malignant urothelial cells that had undergone squamous differentiation. Keratin 6 was shown to consistently stain only cells having some evidence of squamous differentiation. Keratin 16 was shown to follow the staining pattern of keratin 6. The isolates and tumor heterotransplants were all examined for keratin 6, 16 and 17 mRNA and protein expression.

Variation of keratin 7 expression and other phenotypic characteristics of independent isolates of cadmium transformed human urothelial cells (UROtsa).

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd(2+)) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd(2+) transformed cell lines were isolated to determine if independent exposures of the cell line to Cd(2+) would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice, and for the expression of keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to those of the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology, and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd(2+) transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd(2+) transformed cells have both similarities and differences in their phenotype.

Peptidyl-Lys metalloendopeptidase-catalyzed 18O labeling for comparative proteomics: application to cytokine/lipolysaccharide-treated human retinal pigment epithelium cell line.

We recently proposed a comparative proteomic method utilizing proteolytic 18O labeling of peptides catalyzed by peptidyl-Lys metalloendopeptidase (Lys-N) (Rao, K. C. S., Carruth, R. T., and Miyagi, M. (2005) Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics. J. Proteome Res. 4, 507-514). Unlike trypsin, which generates a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species, Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide in H2(18)O solvent. This study reports the first biological application of the Lys-N-based proteolytic 18O labeling method, characterizing the proteome changes of cytokine/lipopolysaccharide-treated verses untreated human retinal pigment epithelium (ARPE-19) cells. The study resulted not only in the identification of 584 proteins but also the determination of the relative abundances of 562 proteins in the two proteomes. The results demonstrate the usefulness of the Lys-N-based proteolytic 18O labeling method in comparative proteomic studies. The results also provide the most comprehensive description of the retinal pigment epithelium proteome to date.

Nuclear and plasma membrane localization of SH3BP4 in retinal pigment epithelial cells.

PURPOSE: The SH3BP4 protein contains domains belonging to the Eps15-Homology (EH) network family of endocytosis proteins and a C-terminal death domain. The purpose of this study was to determine the expression of SH3BP4 in ARPE-19, Y79 and COS-7 cell lines and to determine SH3BP4 subcellular localization within ARPE-19 cells. METHODS: A chicken anti-human SH3BP4 antibody was generated that specifically immunostains SH3BP4 fusion proteins and a corresponding endogenous protein band at 120 kDa. Protein expression of SH3BP4 was determined using western analysis of multiple cell lines and dissected retinal tissue. Intracellular localization of both endogenous SH3BP4 and SH3BP4 fusion proteins were determined using subcellular fractionation and microscopy studies using ARPE-19 and COS-7 cells. RESULTS: The retinal pigment epithelial (RPE) cell line ARPE-19 was found to express SH3BP4 protein at more than 7 fold the levels in Y79 retinoblastoma cells and more than 2.5 fold the levels in COS-7 cells. Both the RPE and neural retinal layers of the eye were also found to express the SH3BP4 protein. SH3BP4 endogenous and fusion proteins were found to localize to both membrane and nuclear fractions but not the cytosol in subcellular fractionation experiments. Subsequent microscopy analyses show that SH3BP4 fusion proteins localize to the plasma membrane and the nuclear periphery. CONCLUSIONS: These studies show that SH3BP4 is expressed in the RPE and neural retina in vivo, and in ARPE-19, Y79, and COS-7 cell lines. Compared to other EH network and death domain proteins, SH3BP4 fusion proteins have an unusual intracellular localization to the plasma membrane and the nuclear periphery. The present demonstration of the suborganelle localization in conjunction with the unique domain combinations belonging to both endocytosis and cell death pathways suggests that SH3BP4 has physiological significance for RPE cells.

Interaction of lumican with aggrecan in the aging human sclera.

PURPOSE: Lumican is a keratan sulfate proteoglycan originally identified in cornea, but present in a variety of connective tissues where it presumably regulates collagen fibril formation and organization. The present study was designed to describe the chemical nature of lumican core protein in the aging human sclera. METHODS: Western blot analyses, immunohistochemistry, and immunoaffinity chromatography were used to detect and purify the lumican core protein from tissue extracts from human donors 6 to 89 years of age. Treatment of lumican with chondroitinase ABC, keratanase-I and -II, and/or endo-beta-galactosidase was used to determine the degree of glycosylation of the lumican core protein. RESULTS: Lumican was present in the human sclera as a 70- to 80-kDa core protein with short unsulfated lactosaminoglycan side chains. In addition, on Western blots, a larger >200-kDa species was apparent that was immunologically related to lumican. This high-molecular-weight material increased in scleral extracts with increasing age. The complex was most abundant in unreduced samples, and approximately two thirds of the 70- to 80-kDa lumican core protein was released from the complex on reduction of the scleral extract. Further characterization of the >200-kDa lumican-immunopurified complex indicated that aggrecan (the cartilage proteoglycan) was covalently associated with lumican. CONCLUSIONS: Reducible and nonreducible lumican-aggrecan interactions occur in the scleral extracellular matrix and result in the formation of high-molecular-weight complexes that increase with age. These results represent the first report demonstrating lumican-aggrecan interactions and suggest they may play a role in age-related scleral extracellular matrix changes.