Proteoglycan (Keratan Sulfate) Barrier in Developing Human Forebrain Isolates Cortical Epileptic Networks From Deep Heterotopia, Insulates Axonal Fascicles, and Explains Why Axosomatic Synapses Are Inhibitory.

Axons from deep heterotopia do not extend through U-fibers, except transmantle dysplasias. Keratan sulfate (KS) in fetal spinal cord/brainstem median septum selectively repels glutamatergic axons while enabling GABAergic commissural axons. Immunocytochemical demonstration of KS in neocortical resections and forebrain at autopsy was studied in 12 fetuses and neonates 9-41 weeks gestational age (GA), 9 infants, children, and adolescents and 5 patients with focal cortical dysplasias (FCD1a). From 9 to 15 weeks GA, no KS is seen in the cortical plate; 19-week GA reactivity is detected in the molecular zone. By 28 weeks GA, patchy granulofilamentous reactivity appears in extracellular matrix and adheres to neuronal somata with increasing intensity in deep cortex and U-fibers at term. Perifascicular KS surrounds axonal bundles of both limbs of the internal capsule and within basal ganglia from 9 weeks GA. Thalamus and globus pallidus exhibit intense astrocytic reactivity from 9 weeks GA. In FCD1a, U-fiber reactivity is normal, discontinuous or radial. Ultrastructural correlates were not demonstrated; KS is not electron-dense. Proteoglycan barrier of the U-fiber layer impedes participation of deep heterotopia in cortical epileptic networks. Perifascicular KS prevents aberrant axonal exit from or entry into long and short tracts. KS adhesion to neuronal somatic membranes may explain inhibitory axosomatic synapses.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells.

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell-microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 µM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities of SLRP in response to CSC enrichment, simultaneously acquiring TMZ resistance, cellular heterogeneity, and a quiescent phenotype, suggesting a novel pivotal role for SLRP in drug resistance and cell plasticity of CSC-like, allowing cell survival and ECM/niche modulation potential.

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance]. / Proteinas inhibidoras de la regeneracion neuritica en la matriz extracelular: estructura, interacciones moleculares y sus funciones. Mecanismos del balance extracelular.

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

Extracellular matrix protein lumican regulates inflammation in a mouse model of colitis.

BACKGROUND: Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen-associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by Toll-like receptor (TLR) 4, and neutrophil chemotaxis via integrins. METHODS: Here we investigated the role of lumican in the development of colitis mediated by intrarectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum(+/+) and Lum(-/-) mice. RESULTS: The TNBS treated Lum(+/+) mouse colons showed marked increases in CXCL1, tumor necrosis factor alpha (TNF-α), and neutrophil infiltration, whereas these responses were significantly dampened in the Lum(-/-) mice. The nuclear factor kappa B (NF-κB) transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum(+/+) but not in Lum(-/-) colons. Also, nuclear translocation of NF-κB was delayed in LPS stimulated Lum(-/-) primary peritoneal macrophages. CONCLUSIONS: The Lum(-/-) mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4(-/-) and MyD88(-/-) mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

Keratan sulfate suppresses cartilage damage and ameliorates inflammation in an experimental mice arthritis model.

Proteoglycans bearing keratan sulfate (KS), such as aggrecan, are components of the human cartilage extracellular matrix (ECM). However, the role of KS in influencing cartilage degradation associated with arthritis remains to be completely understood. KS side chains of the length found in human cartilage are not found in murine skeletal tissues. Using a murine model of inflammatory polyarthritis and cartilage explants exposed to interleukin-1α (IL-1α), we examined whether administering KS could influence intraarticular inflammation and cartilage degradation. Acute arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail, followed by an intraperitoneal injection of lipopolysaccharide. This treatment was followed by an intraperitoneal KS administration in half of the total mice to evaluate the therapeutic potential of KS for ameliorating arthritis. To investigate the therapeutic potential ex vivo, we examined cartilage fragility by measuring IL-1α-induced aggrecan release from cartilage explants treated with or without KS. Intraperitoneal KS administration ameliorated arthritis in DBA/1J mice. The aggrecan release induced by IL-1α was less in cartilage explants containing media with KS than in those without KS. Our data indicate that exogenous KS ameliorated arthritis in vivo and suppressed cartilage degradation ex vivo. KS may have important therapeutic potential in the treatment of inflammatory arthritis. The mechanism responsible for this requires further investigation, but KS may become a novel therapeutic agent for treating inflammatory diseases such as rheumatoid arthritis.

Impaired skin wound healing in lumican-null mice.

BACKGROUND: Previous studies have demonstrated that the lack of lumican delayed corneal wound healing in lumican-null (Lum(-/-) ) mice. This defect is rescued by the addition of glycosylated lumican core protein to the injured corneas. OBJECTIVES: We examined the hypothesis that lumican is also required for the healing of cutaneous wounds using Lum(-/-) mice. METHODS: We demonstrated the basic thinner skin phenotypes in Lum(-/-) mice at different time points and the changes in arrangement of collagen fibres by transmission electron microscopy (TEM). A full skin thickness wound was generated by punch biopsy (6 mm diameter) in experimental Lum(-/-) and wild-type mice. The closure of injured skin was measured after various periods of time (3, 6, 12, 18 days). Specimens of injured and uninjured skin (serving as control) were then subjected to morphological examination with haematoxylin and eosin and Masson trichrome stains, and by TEM. Immunohistochemical staining with anti-CD68 antibody was used to assess the presence of macrophages in injured skin healing for various periods of time. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to elucidate the transforming growth factor (TGF)-ß1-induced myofibroblast phenotypic genes. RESULTS: Skin of adult Lum(-/-) mice (3 months and older) was much thinner (40% less) than that of age-matched wild-type mice. This phenomenon was aggravated in older mice. TEM revealed disoriented and irregular collagen fibrils in the dermis of Lum(-/-) mice. Delayed wound healing with an increase in inflammatory macrophages was compatible with the delayed response of the expression of TGF-ß1, type I collagen α1 and fibronectin at the mRNA level by semiquantitative RT-PCR in the Lum(-/-) mice. CONCLUSIONS: Our data demonstrate that lumican plays pivotal roles in skin collagen fibrillogenesis and wound healing.

Enhanced expression of lumican inhibited the attachment and growth of human embryonic kidney 293 cells.

Lumican is a member of a small leucine-rich proteoglycan (SLRP) family and it regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. Lumican expression was reported in various kinds of tumor cells. Lumican inhibits the growth of melanoma cells, but the lumican in pancreatic cancer correlated with an advanced stage and retroperitoneal and duodenal invasion. In this study, we clarified whether the enhanced expression of lumican contributes to cellular attachment, growth, colony formation, migration and invasion. HEK 293 cell, stably transfected with lumican cDNA synthesized and secreted a 50 kDa lumican protein at high levels in culture medium. The cells showed a polygonal appearance with long projections and the degree of adhesion of the cells to fibronectin was lower than that of empty vector transfected control cells (mock cells). In contrast, the degree of adhesion of the cells to type I collagen was not different from that of mock cells. The expression levels of alpha5 integrin, the major integrin subunit for fibronectin, were lower in lumican-transfected HEK cells than in mock cells. Furthermore, lumican-transfected HEK cells showed reduced growth rates in vitro and did not form colonies in soft agar. Phosphorylation of AKT, extracellular signal-regulated kinase (ERK) 1/2 and mammalian target of rapamycin (mTOR) decreased in the lumican-transfected HEK cells. Cell migration and invasion were not altered in lumican-transfected HEK cells and mock cells. These findings indicate that the 50kDa lumican protein plays important roles in the inhibition of HEK cell attachment and growth, and it might inhibit the activation of integrin pathways.

Collagen fibril organization in the pregnant endometrium of decorin-deficient mice.

In the pregnant mouse endometrium, collagen fibrillogenesis is characterized by the presence of very thick collagen fibrils which are topographically located exclusively within the decidualized stroma. This dynamic biological process is in part regulated by the small leucine-rich proteoglycans decorin and biglycan. In the present study we utilized wild-type (Dcn(+/+)) and decorin-deficient (Dcn(-/-)) time-pregnant mice to investigate the evolution of non-decidualized and decidualized collagen matrix in the uterine wall of these animals. Ultrastructural and morphometric analyses revealed that the organization of collagen fibrils in the pregnant endometrium of both non-decidualized and decidualized stroma showed a great variability of shape and size, regardless of the genotype. However, the decidualized endometrium from Dcn(-/-) mice contained fibrils with larger diameter and more irregular contours as compared to the wild-type littermates. In the Dcn(-/-) animals, the proportion of thin (10-50 nm) fibrils was also higher as compared to Dcn(+/+) animals. On day 7 of pregnancy, biglycan was similarly localized in the decidualized endometrium in both genotypes. Lumican immunostaining was intense both in decidualized and non-decidualized stroma from Dcn(-/-) animals. The present results support previous findings suggesting that decorin participates in uterine collagen fibrillogenesis. In addition, we suggest that the absence of decorin disturbs the process of lateral assembly of thin fibrils, resulting in very thick collagen fibrils with irregular profiles. Our data further suggest that decorin, biglycan and lumican might play an interactive role in collagen fibrillogenesis in the mouse endometrium, a process modulated according to the stage of pregnancy.

Lumican, a small leucine-rich proteoglycan.

Lumican belongs to the family of small leucine-rich repeat proteoglycans. Recent studies have shown that lumican participates in the maintenance of tissue homeostasis and modulates cellular functions including cell proliferation, migration, and differentiation. The expression of lumican has been correlated to the growth and metastasis of various malignancies; however, its exact role in tumorogenesis remains elusive. This review focuses upon the role of lumican in cell biology, providing insights into molecular mechanisms that lumican likely utilizes to control processes relevant to tumorogenesis.

Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells.

Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine-rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG-63) and well-differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real-time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non-metastatic, well-differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG-63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down-regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG-63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.

Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer.

Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in carcinoid tumor, breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we aimed to clarify the role of lumican in pancreatic cancer. Reverse-transcription polymerase chain reaction and Western blot analyses revealed lumican mRNA and protein expression in six pancreatic ductal adenocarcinoma cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1, Capan-1, PK-1 and PK-8). On the basis of its immunoreactivity, lumican was found to be localized in islet cells of normal pancreatic tissues, but not in exocrine cells. In pancreatic cancer tissues, lumican was predominantly localized in the cytoplasm of cancer cells in 30 out of 53 (56.6%) cancer patients, whereas lumican was detected in stromal tissues in 36 out of 53 (67.9%) cancer patients. Lumican expression in pancreatic cancer cells did not correlate with clinicopathological factors, whereas lumican expression in stromal tissues correlated with the female gender, advanced stage, retroperitoneal and duodenal invasion and residual tumor (p=0.030, 0.038, 0.049, 0.049 and 0.048, respectively). Patients with lumican-positive cancer cells tended to survive longer than those with lumican-negative cancer cells (p=0.286), but patients with lumican-positive stromal tissues had shorter survival than those with lumican-negative stromal tissues (p=0.062). These results suggest that lumican in stromal tissues plays an important role in the growth and invasion of pancreatic cancer.

Role of the small leucine-rich proteoglycan (SLRP) family in pathological lesions and cancer cell growth.

The roles of lumican, a member of the small-leucine-rich-proteoglycan (SLRP) family, in pathological fibrosis, cancer tissues and tumor cell growth were reviewed. Lumican is predominantly localized in the areas of pathological fibrosis including the thickened intima of human coronary arteries, ischemic and reperfused hearts, and acute pancreatitis and chronic pancreatitis (CP)-like lesions adjacent to pancreatic cancer nests. In these lesions, lumican mRNA and protein were transiently and ectopically overexpressed in most of the vascular smooth muscle cells (VSMCs) that migrated into the thickened intima, myocardial cells adjacent to an ischemic lesion, acinar cells, islet cells and fibroblasts of pathological pancreatic tissues. The low expression level of lumican in breast cancer is associated with rapid progression and poor survival. Lumican mRNA in breast cancer is overexpressed in fibroblasts adjacent to cancer cells but not in cancer cells. Furthermore, the high expression level of lumican is associated with a high pathological tumor grade, a low estrogen receptor level in the cancer tissues, and young age of patients. The suppression of lumican expression in culture cells induces their cell growth. Lumican-transfected tumor cells are characterized by a strong suppression of their anchorage-independent growth and capacity of invasion. Lumican significantly suppressed subcutaneous tumor formation in syngenic mice, with a concomitant decrease in cyclin D1 expression level, and induced and/or enhanced the apoptosis of these cells. The autocrine mechanism in cancer cells and the paracrine mechanism in cancer cells and fibroblasts via transforming growth factor (TGF)-beta and Smad signals may play important roles in the regulation of tumor growth by SLRPs.

Ultrastructural and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans.

Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.

Lumican regulates corneal inflammatory responses by modulating Fas-Fas ligand signaling.

PURPOSE: The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling. The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries. METHODS: Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts. Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively. Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA. RESULTS: Lum-/- -injured corneas showed significantly lower caspase-3/7 activity (apoptosis). Lum-/- -wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta. The Fas protein level, before and after wounding, was dramatically lower in Lum-/- - compared with Lum+/+-injured cornea. However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level. Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican. CONCLUSIONS: The data suggest that lumican binds FasL and facilitates induction of Fas. Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing.

The small leucine-rich proteoglycan lumican inhibits melanoma progression.

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.

Lumican suppresses cell proliferation and aids Fas-Fas ligand mediated apoptosis: implications in the cornea.

Lumican, an extracellular matrix (ECM) keratan sulfate proteoglycan, binds fibrillar collagen and limits collagen fibril diameter in the cornea, skin and tendon. Lumican-deficient mice (Lum(-/-)) develop abnormally thick collagen fibrils, translucent corneas and fragilities of the skin and the tendon. In addition to modulating interstitial ECM structure, here we hypothesized that lumican regulates proliferation and apoptosis of cells residing in the interstitium. Corneal and embryonic fibroblasts from the Lum(-/-) mouse show increased growth in culture. Lum(-/-) mouse embryonic fibroblasts (MEF), compared to their wild type counterparts, display increased rates of proliferation and decreased apoptosis. Ectopic expression of lumican in Lum(-/-) MEF or exogenous recombinant lumican in the culture medium reduces proliferation to rates seen in the Lum(+/+) MEF. We further investigated the implications of lumican's proliferation and apoptosis regulatory role in the cornea where lumican is a major component of the stromal matrix. Stromal keratocytes undergo proliferation and apoptosis during corneal maturation and in the healing of injured cornea. The Lum(-/-) mouse shows increased proliferation and decreased apoptosis of stromal keratocytes during postnatal corneal maturation at the 10-day age. Apoptosis is also significantly down regulated in Lum(-/-) vis-à-vis Lum(+/+) mice during stromal wound healing in the adult 6-week age. Lumican appears to regulate these cellular functions by modulating specific cell growth and apoptosis mediators. Thus, Lum(-/-) MEF have decreased p21(WAF1/CIP1), a universal inhibitor of cyclin-dependent kinases and a consequent increase in cyclins A, D1 and E. Furthermore, the tumor suppressor p53, an upstream regulator of p21 is down regulated in the MEF and the cornea of Lum(-/-) mice. The evidence suggests regulation of p21 by lumican in a p53-dependent way. The MEF and the cornea of Lum(-/-) mice also show a dramatic decrease in Fas (CD95). The Lum(-/-) MEF fail to induce Fas upon treatment with Fas ligand. Fas-Fas ligand interaction is an initiating event in apoptosis and its disruption in lumican-deficiency may partly explain the observed decrease in apoptosis. Fas-Fas ligand mediated apoptosis is critical for maintaining the immune privileged status of the cornea, which implies a new and exciting role for lumican in the cornea.

Effect of morpholino antisense oligonucleotide against lumican mRNA in human embryonic kidney (HEK) 293 cells.

Lumican is a member of the small-leucine-rich proteoglycan (SLRP) family and is overexpressed during wound healing of the cornea, in ischemic and reperfused heart, and in several cancer tissues. Lumican is considered to regulate the collagen fibril diameter and interfibrillar spacing. However, the effect of lumican on cell growth has not been adequately examined. In the present study, we attempted to clarify whether lumican contributes to human embryonic kidney (HEK) 293 cell growth, using the morpholino antisense oligonucleotide (m-anti oligo) against lumican mRNA. M-anti oligo is a novel oligonucleotide and exhibits a higher antisense activity, higher water solubility, and greater resistance to nucleases in target cells than phosphorothioate types of oligonucleotide. After delivery of m-anti oligo against lumican mRNA, the fluorescein 5-isothiocyanate (FITC) conjugated oligonucleotides were observed in the cytoplasm and nucleus of HEK 293 cells at 24 h by confocal laser microscopy. M-anti oligo for lumican mRNA strongly inhibited the synthesis of lumican protein in the HEK 293 cells, and the HEK cell growth rate was higher than those in the control groups. These findings may indicate that lumican protein has an inhibitory effect on HEK 293 cell growth in vitro.

Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition.

PURPOSE: Lens epithelial cells (LECs) undergo epithelial-mesenchcymal transition (EMT) after injury and transform into myofibroblasts positive for alpha-smooth muscle actin (alphaSMA), an established marker of this process. Lumican is a keratan sulfate proteoglycan core protein. This study was conducted to examine whether human and mouse LECs express lumican after injury. To determine whether lumican may modulate EMT of LECs in response to injury or to exposure to transforming growth factor-beta2 (TGFbeta2), alphaSMA expression by the LECs was examined in lumican (Lum)-knockout mice in vivo and in organ culture. METHODS: Human postoperative capsular specimens and healing, injured mouse lenses at various intervals were immunostained for lumican or alphaSMA. alphaSMA was also immunolocalized in healing, injured lenses of Lum-knockout mice. Finally, expression of lumican and alphaSMA was examined in lenses of Lum-knockout mice incubated with TGFbeta2. RESULTS: Lumican was immunolocalized in matrix in human postoperative capsular opacification. Lumican and alphaSMA were upregulated in mouse LECs from 8 hours and day 5 after an injury, respectively. LECs accumulated adjacent to the capsular break were of epithelial shape in Lum(-/-) mice and fibroblast-like in Lum(+/-) mice during healing. alphaSMA expression by LECs was significantly delayed in Lum(-/-) mice, indicating that lumican may modulate injury-induced EMT in LECs. TGFbeta2-induced EMT appeared to be suppressed in organ-cultured lenses of Lum(-/-) mice compared with those of Lum(+/+) mice. CONCLUSIONS: Human capsular opacification contains lumican, and mouse LECs upregulate lumican and alphaSMA in response to injury. Loss of lumican perturbs EMT of mouse LECs.

Chondroitin and keratan sulfates have opposing effects on attachment and outgrowth of ventral mesencephalic explants in culture.

During rat brain development, striatal proteoglycan (PG) expression shows specific spatio-temporal modifications suggesting a possible role in the guidance of its dopaminergic afferents. The effects of individual glycosaminoglycans (GAGs) on dopaminergic (DA) neuronal adhesion and outgrowth were therefore studied. We tested the behavior of dissociated embryonic rat mesencephalic cells cultivated on substrate-bound GAGs. Neuronal attachment was very limited and quantitative morphometry revealed variations in DA fiber outgrowth depending on the type and the concentration of GAG used. Next, we developed a cryoculture system to examine how neurons react toward GAGs expressed in situ. Rat brain slices from different developmental stages were used as substrates for embryonic mesencephalic explants. Preferential regions of adherence and outgrowth were observed: the striatum was found to be the most permissive, whereas the cortex was inhibitory. Western blotting experiments confirmed quantitative and qualitative changes in chondroitin sulfate (neurocan, phosphacan) and keratan sulfate (KS) containing PGs in these substrates and enzymatic digestion of GAGs before cryoculture revealed a substantial involvement of PGs in DA neuron adhesion and outgrowth. In particular, CSPGs seemed to mediate the permissive effect of the striatum, whereas KS confers an inhibitory effect to the cortex. PGs may thus be important for limiting midbrain projections to the striatum during development and for maintaining topography in the adult.

Roles of lumican and keratocan on corneal transparency.

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis.