Collagen XVIII regulates extracellular matrix integrity in the developing nephrons and impacts nephron progenitor cell behavior.

Renal development is a complex process in which two major processes, tubular branching and nephron development, regulate each other reciprocally. Our previous findings have indicated that collagen XVIII (ColXVIII), an extracellular matrix protein, affects the renal branching morphogenesis. We investigate here the role of ColXVIII in nephron formation and the behavior of nephron progenitor cells (NPCs) using isoform-specific ColXVIII knockout mice. The results show that the short ColXVIII isoform predominates in the early epithelialized nephron structures whereas the two longer isoforms are expressed only in the later phases of glomerular formation. Meanwhile, electron microscopy showed that the ColXVIII mutant embryonic kidneys have ultrastructural defects at least from embryonic day 16.5 onwards. Similar structural defects had previously been observed in adult ColXVIII-deficient mice, indicating a congenital origin. The lack of ColXVIII led to a reduced NPC population in which changes in NPC proliferation and maintenance and in macrophage influx were perceived to play a role. The changes in NPC behavior in turn led to notably reduced overall nephron formation. In conclusion, the results show that ColXVIII has multiple roles in renal development, both in ureteric branching and in NPC behavior.

Targeting collagen XVIII improves the efficiency of ErbB inhibitors in breast cancer models.

The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.

Temporally and spatially regulated collagen XVIII isoforms are involved in ureteric tree development via the TSP1-like domain.

Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development.

Upregulated integrin α11 in the stroma of cutaneous squamous cell carcinoma promotes skin carcinogenesis.

Integrin α11ß1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11-/- ) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFß1 and collagen crosslinking lysyl oxidases in the Itga11-/- tumor stroma. In summary, our data suggest that α11ß1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11ß1 in skin tumorigenesis.

Deletion of Col15a1 Modulates the Tumour Extracellular Matrix and Leads to Increased Tumour Growth in the MMTV-PyMT Mouse Mammary Carcinoma Model.

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.

Collagen XVIII in tissue homeostasis and dysregulation - Lessons learned from model organisms and human patients.

Collagen XVIII is a ubiquitous basement membrane (BM) proteoglycan produced in three tissue-specific isoforms that differ in their N-terminal non-collagenous sequences, but share collagenous and C-terminal non-collagenous domains. The collagenous domain provides flexibility to the large collagen XVIII molecules on account of multiple interruptions in collagenous sequences. Each isoform has a complex multi-domain structure that endows it with an ability to perform various biological functions. The long isoform contains a frizzled-like (Fz) domain with Wnt-inhibiting activity and a unique domain of unknown function (DUF959), which is also present in the medium isoform. All three isoforms share an N-terminal laminin-G-like/thrombospondin-1 sequence whose specific functions still remain unconfirmed. The proteoglycan nature of the isoforms further increases the functional diversity of collagen XVIII. An anti-angiogenic domain termed endostatin resides in the C-terminus of collagen XVIII and is proteolytically cleaved from the parental molecule during the BM breakdown for example in the process of tumour progression. Recombinant endostatin can efficiently reduce tumour angiogenesis and growth in experimental models by inhibiting endothelial cell migration and proliferation or by inducing their death, but its efficacy against human cancers is still a subject of debate. Mutations in the COL18A1 gene result in Knobloch syndrome, a genetic disorder characterised mainly by severe eye defects and encephalocele and, occasionally, other symptoms. Studies with gene-modified mice have elucidated some aspects of this rare disease, highlighting in particular the importance of collagen XVIII in the development of the eye. Research with model organisms have also helped in determining other structural and biological functions of collagen XVIII, such as its requirement in the maintenance of BM integrity and its emerging roles in regulating cell survival, stem or progenitor cell maintenance and differentiation and inflammation. In this review, we summarise current knowledge on the properties and endogenous functions of collagen XVIII in normal situations and tissue dysregulation. When data is available, we discuss the functions of the distinct isoforms and their specific domains.

Endostatin and transglutaminase 2 are involved in fibrosis of the aging kidney.

Endostatin (EST), an antiangiogenic factor, is enriched in aging kidneys. EST is also an interactive partner of transglutaminase 2 (TG2), an enzyme that cross-links extracellular matrix proteins. Here we tested whether EST and TG2 play a role in the fibrosis of aging. In wild-type mice, aging kidneys exhibited a 2- to 4-fold increase in TG2 paralleled by increased cross-linked extracellular matrix proteins and fibrosis. Mice transgenic to express EST showed renal fibrosis at a young age. One-month delivery of EST via minipumps to young mice showed increased renal fibrosis that became more robust when superimposed on folic acid-induced nephropathy. Upregulated TG2 and impaired renal function were apparent with EST delivery combined with folic acid-induced nephropathy. Subcapsular injection of TG2 and/or EST into kidneys of young mice not only induced interstitial fibrosis, but also increased the proportion of senescent cells. Thus, kidney fibrosis in aging may represent a natural outcome of upregulated EST and TG2, but more likely it appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, EST and TG2.

Lysyl hydroxylase 3 modifies lysine residues to facilitate oligomerization of mannan-binding lectin.

Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers.

The activities of lysyl hydroxylase 3 (LH3) regulate the amount and oligomerization status of adiponectin.

Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.

Serum adipokine levels in adults with a history of childhood maltreatment.

Individuals with a history of childhood maltreatment present increased rates of metabolic disturbances, but the underlying mechanisms for such phenomena are poorly understood. This study examined whether the secretion of adipokines, adipocyte-derived inflammation markers closely associated with metabolic disorders, is altered in individuals with a history of childhood maltreatment. The serum levels of inflammatory markers adiponectin and resistin were measured from 147 general population participants who had a history of adverse mental symptoms, and who also reported their experiences of childhood maltreatment. Participants with experiences of childhood maltreatment (n=30) had lowered levels of serum adiponectin (p=0.007) and resistin (p=0.028). The differences in adiponectin levels persisted in multivariate modeling with adjustments for age, gender, and body mass index (OR for each 1 standard deviation decrease in the serum adiponectin level 2.65, 95% CI 1.31-5.35, p=0.007). Additional adjustments for marital status or a diagnosis of major depressive disorder, or the exclusion of individuals using NSAIDs, oral corticosteroids, or antidepressants did not alter the results. The association between resistin levels and childhood maltreatment did not remain independent in the same models. Our findings suggest that in individuals with previously reported adverse mental symptoms, a history of childhood maltreatment is independently associated with lowered levels of the anti-inflammatory marker adiponectin. This may lead to a lowered anti-inflammatory buffer capacity, which can, in turn, increase the susceptibility to physical and psychological states characterized by pronounced pro-inflammation.

Serum IL-7 and G-CSF in major depressive disorder.

Major depressive disorder (MDD) has been associated with dysregulated immune systems and impaired T cell function, but data on depression-related alterations in the levels of immunomodulatory growth factors are scarce. In order to further clarify the mechanisms underlying immune system dysregulation in depressed subjects, we examined the associations between MDD and serum levels of two immunomodulatory growth factors, interleukin (IL)-7 and granulocyte-colony stimulating factor (G-CSF), in 122 subjects (MDD with long-term symptomatology, n=61; controls, n=61). The MDD subjects had lowered levels of IL-7. In a model adjusted for age, gender and body mass index, subjects in the lowest tertile of IL-7 had a 3.4-fold increased likelihood for MDD (p=0.010). Further adjustments for sleep disturbances, alcohol use, smoking, and metabolic syndrome did not alter these findings. Moreover, the exclusion of subjects with rheumatoid arthritis, coronary heart disease, or the use of non-steroidal anti-inflammatory medications or oral corticosteroids only slightly attenuated the findings. The G-CSF levels did not differ between the two groups. The lowering of the serum levels of IL-7, a regulator of T cell homeostasis, in MDD subjects may underlie the depression-related impaired T cell function.

Genetic and environmental determinants of total and high-molecular weight adiponectin in families with low HDL-cholesterol and early onset coronary heart disease.

OBJECTIVE: Plasma adiponectin and high-density lipoprotein cholesterol (HDL-C) exhibit a well-known positive metabolic correlation. Neither heritability nor genome-wide linkage analysis for the high-molecular weight (HMW) adiponectin is available. This work estimates the genetic and environmental determinants and the heritabilities of the adiponectins and lipid traits in Finnish families with early onset coronary heart disease (CHD) and low HDL-C. METHODS: Heritability and genome-wide univariate linkage analysis was performed for total and HMW adiponectin in extended families from Northern Finland with early onset CHD and low HDL-C using a variance components approach. The genetic and environmental correlations between the plasma adiponectins and various lipid traits were also studied and a bivariate analysis for HDL-C and the adiponectins carried out. RESULTS: In the partial correlation analysis (adjusted for sex, age, BMI and statin use) the adiponectins showed a stronger correlation with HDL-C (total 0.57, p=0.001, HMW 0.51, p<0.005) than with any other lipid trait in unrelated subjects. Our estimates detected strong heritability for total (0.53+/-0.10), HMW (0.51+/-0.10) and the HMW/total adiponectin ratio (0.68+/-0.11). Univariate linkage analysis showed suggestive evidence of linkage on chromosome 11p15 for total adiponectin and on 3q13.2-q24 and 6p21 for the HMW adiponectin. The strongest environmental cross-correlation between the adiponectins and lipids was seen between HDL-C and total adiponectin (rhoe=0.64, p<0.05), whereas the strongest genetic correlation was detected between low-density lipoprotein cholesterol and the HMW adiponectin (rhog=-0.48, p<0.05). CONCLUSION: No significant genetic correlations between HDL-C and the adiponectins were observed. Therefore, the metabolic association between HDL-C and adiponectin is most likely regulated by complex genetic pathways and environmental factors.

Serum chemokine levels in major depressive disorder.

OBJECTIVE: To examine the role of chemokines of two major chemokine families, CC and CXC, in major depressive disorder (MDD) in a population-based sample. METHOD: The serum levels of CC chemokines MCP-1 and MIP-1beta, and CXC chemokine IL-8 were measured from 122 participants (MDD group, n=61; controls, n=61). Depression severity was assessed with the 29-item Hamilton Depression Rating Scale. RESULTS: The MDD group had lower levels of MCP-1, MIP-1beta and IL-8 than the healthy controls. The likelihood of major depressive disorder for participants with chemokine levels below the median (MCP-1: < 26.26 pg/mL; MIP-1beta: < 42.57 pg/mL; IL-8: < 2.86 pg/mL) was 3.6 (p=0.002) for MIP-1beta and 2.4 (p=0.037) for IL-8 in regression models adjusted for age, gender, body mass index, smoking, and alcohol consumption. MCP-1 did not associate with the presence of MDD after adjustments for potential confounders. Further adjustments for somatic illnesses or medications did not affect these findings. CONCLUSION: Our findings suggest that depression-related alterations of inflammatory markers may be more complex than previously assumed, and that at least some of the chemokines may be down-regulated.

Reduction of lysyl hydroxylase 3 causes deleterious changes in the deposition and organization of extracellular matrix.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF(+/-)) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF(+/-), may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.

Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines.

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. These hydroxylysine-linked carbohydrates were shown recently to be indispensable for the formation of basement membranes (Ruotsalainen, H., Sipilä, L., Vapola, M., Sormunen, R., Salo, A. M., Uitto, L., Mercer, D. K., Robins, S. P., Risteli, M., Aszodi, A., Fässler, R., and Myllylä, R. (2006) J. Cell Sci. 119, 625-635). Analysis of LH3 knock-out embryos and cells in this work indicated that loss of glycosylated hydroxylysines prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens. Mice lacking the LH activity of LH3 produced slightly underglycosylated type IV and VI collagens with abnormal distribution. The altered distribution and aggregation of type VI collagen led to similar ultrastructural alterations in muscle to those detected in collagen VI knockout and some Ullrich congenital muscular dystrophy patients. Our results provide new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion, assembly, and distribution of highly glycosylated collagen types.

Expanding the lysyl hydroxylase toolbox: new insights into the localization and activities of lysyl hydroxylase 3 (LH3).

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling.

The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult.

Lysyl hydroxylase catalyzes the hydroxylation of lysine residues in collagenous sequences. Three isoforms (LH1, LH2 and LH3) of lysyl hydroxylase have been characterized, and LH2 is present as two alternatively spliced forms. In order to better understand the functional differences between the isoforms in vivo, the expression of the different isoforms was studied in mouse embryos and adult tissues. Our data indicate a widespread expression of all isoforms during embryogenesis, whereas the expression profiles become more specialized in adult tissues. The expression of LH2 was more tissue-specific, whereas a uniform and housekeeping like behavior was observed for LH3. Some cells express both LH2 and LH3, while a clear cell specificity was seen in some tissues. Moreover, immunoelectron microscopy revealed differences in the localization of LH2 and LH3. LH2 was localized intracellularly in the ER in all tissues studied, whereas the localization of LH3 was either intracellular or extracellular or both, depending on the tissue. Furthermore, our data indicate that the alternative splicing of LH2 is developmentally regulated. The short form of LH2 (LH2a) is the predominant form until E11.5; the long form (LH2b) dominates thereafter and is the major form in many adult tissues. Interestingly, however, adult mouse kidney and testis express exclusively the short form, LH2a. The results reveal a specific regulation for the expression of LH isoforms as well as for alternative splicing of LH2 during embryogenesis and in different tissues.

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling.

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.

Glycosylation catalyzed by lysyl hydroxylase 3 is essential for basement membranes.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.