Connective Tissue Integrity Research Articles

Effect of Mechanical Strain on the Collagen VI Pericellular Matrix in Anterior Cruciate Ligament Fibroblasts

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.

Pulmonary artery rupture during Swan-Ganz catheterisation: A case report

Catheter-induced pulmonary artery rupture is an infrequent complication that may occur during invasive cardiopulmonary monitoring. Fatal cases are uncommon and result from hemoptysis and flooding of the opposite lung with resulting hypoyxia. Alpha-1-antitrypsin deficiency is a rare genetic disorder characterised by low serum levels of alpha-1-antitrypsin, critical in maintaining connective tissue integrity. Besides pulmonary emphysema, recent observations suggest that alpha-1-antitrypsin deficiency may also be involved in vascular wall weakening, thereby predisposing arteries to dissection and aneurysm formation. In this article, we describe an autopsy case of pulmonary artery iatrogenic rupture due to insertion of a Swan-Ganz catheter in an 82-year-old woman suffering from pulmonary hypertension and alpha-1-antitrypsin deficiency. The exact source of bleeding could not be precisely identified during autopsy due to the extent of tissue hemorrhage, though postmortem angiography revealed a contrast medium extravasation from a branch of the left pulmonary lower lobar artery. The case herein emphasises the importance of postmortem angiography in facilitating the detection of vascular injuries, the importance of familiarity with intensive care techniques and procedures on behalf of forensic pathologists as well as in-depth knowledge of all possible contributing conditions and predisposing disorders in the pathogenesis of death.

Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a(-/-) and Mep1b(-/-) mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

진피섬유모세포에서 대복피추출물의 세포외기질 합성 촉진 효과

Dermal fibroblasts produce the many components of the extracellular matrix (ECM) that are needed to maintain connective tissue integrity and repair tissue injuries. This study investigated the effects of Areca catechu extract (ACE) on dermal fibroblast cell activation. Cultured human dermal fibroblasts were treated with ACE, and then ECM production was determined by ELISA, Western blot and RT-PCR. ACE significantly accelerated the production of type 1 collagen, fibronectin, and transforming growth factor (TGF)- by ELISA and type 1 collagen by Western blot assay. ACE also increased the gene expression of COL1A1, TGF-, keratinocyte growth factor (KGF) and insulin growth factor (IGF)-1. These results suggest that ACE has the potential to stimulate ECM production and that it might be suitable for maintaining skin texture.

The Relationship between Cortical Blood Flow and Sub-Cortical White-Matter Health across the Adult Age Span

Degeneration of cerebral white matter is commonly observed in aging, and the associated degradation in neural connectivity contributes to cognitive decline in older adults. Vascular dysfunction has been implicated as a potential mechanism for general age-related neural tissue deterioration; however, no prior study has examined the direct relationship between cortical vascular health and subcortical white-matter integrity. In this work, we aimed to determine whether blood supply to the brain is associated with microstructural integrity of connective tissue, and whether such associations are regionally specific and mainly accounted for by aging. We examined the association between cerebral blood flow (CBF) in the cortical mantle, measured using arterial spin labeling (ASL), and subcortical white-matter integrity, measured using diffusion tensor imaging (DTI), in a group of healthy adults spanning early to late adulthood. We found cortical CBF to be significantly associated with white-matter integrity throughout the brain. In addition, these associations were only partially tied to aging, as they remained even when statistically controlling for age, and when restricting the analyses to a young subset of the sample. Furthermore, vascular risk was not a prominent determinant of these effects. These findings suggest that the overall blood supply to the brain is an important indicator of white-matter health in the normal range of variations amongst adults, and that the decline in CBF with advancing age may potentially exacerbate deterioration of the connective anatomy of the brain.

Villous Papillary Thyroid Carcinoma: a Variant Associated with Marfan Syndrome

Marfan syndrome (MFS) is an autosomal dominant hereditary disorder of connective tissue associated with perturbations in transforming growth factor β (TGF-β) biology, most often due to mutations in FBN1 gene that encodes fibrillin-1. To our knowledge, there is no known association of MFS with thyroid carcinoma. We report a 46-year-old man with known history of MFS who developed an unusual histological variant of papillary thyroid carcinoma. The tumor exhibited a widely invasive florid papillary growth pattern with prominent long villous fronds. Immunohistochemical and molecular analysis revealed a BRAF(V600E) mutation, evidence of aggressive biomarker expression (positivity for HBME-1, cytokeratin 19, galectin-3 and cyclin D1, and loss of p27), and changes associated with TGF-β-related epithelial-to-mesenchymal transition with active phospho-SMAD signaling. We introduce a unique histological pattern of papillary thyroid carcinoma that is associated with MFS. The combination of BRAF(V600E) mutation in the setting of altered TGF-β signaling and weak connective tissue integrity associated with MFS may cooperate and possibly be responsible to form this unique villous morphology with epithelial-to-mesenchymal transition and invasive growth.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.

Contemporary management of vascular Ehlers–Danlos syndrome

Vascular Ehlers-Danlos syndrome (EDS) results from mutations in the formation of type III collagen. This leads to various potentially lethal complications including rupture of the arterial vessels, intestinal organs, and the uterus. This review summarizes recent cohort studies that have improved our medical and surgical management of complications associated with vascular EDS. Vascular EDS is associated with a shortened overall survival due to potential complications, namely loss of connective tissue integrity in blood vessels and increased risk of arterial rupture. The traditional approach has been to treat such complications conservatively unless they are life threatening. There have been challenges to this treatment paradigm based on recent reports. Treatment with the beta blocker Celiprolol was shown in a randomized study to be associated with a three-fold decrease in arterial rupture in vascular EDS patients. Furthermore, it was shown by observational studies that elective surgical repair of blood vessels at risk of rupture may be safely undertaken at tertiary referral centers that have expertise in managing connective tissue disorders. Novel approaches using endovascular therapy with coil embolization have also been attempted with good results in the treatment of ruptured pseudoaneurysms, visceral aneurysms, and carotid-cavernous fistulas. New evidence-based treatments have greatly expanded the medical and surgical management options for patients with EDS. These patients are best managed by multidisciplinary teams of interventionalists, cardiologists, and geneticists in tertiary centers with expertise in managing connective tissue disorders.

The effect of cyclic mechanical strain on the expression of adhesion‐related genes by periodontal ligament cells in two‐dimensional culture

Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.

Role of polyphenolic compounds on biological activity of collagenous materials

Collagen, a unique connective tissue protein, is extensively used as biocompatible biomaterial in wound healing [1], cosmetics [2] and tissue engineering [3]. Due to its high sensitivity to enzymatic degradation, cross-linking of collagenous materials must be a compulsory step in their fabrication. Chemical cross-linking agents are able to introduce new covalent bonds in collagen structure, but are cytotoxic. The ability of natural plant polyphenolic compounds to stabilize collagen structure while preserving its cytocompatibility it is now established [4]. This study aimed to investigate the interaction of small molecules from plants with collagen and their effect on its biological properties. Three mixtures of collagen with a polyphenolic extract of Urtica dioica L., a plant-derived flavonoid (quercetin) and a flavin from milk (riboflavin), respectively, were conditioned as porous materials by freeze-drying technique. A collagen-glutaraldehyde mixture was used as control. Free amino groups present in the mixtures were spectrophotometrically assayed. An in vitro experimental model using bacterial collagenase was used to mimic the enzymatic attack on the collagenous materials implanted in vivo. The swelling capacity and in vitro cytocompatibility tested according to ISO 10993–5 on fibroblasts from NCTC cell line were evaluated. Results showed a good correlation between the free amino groups and the biodegradability of each mixture. The values of swelling capacity were at least 70-fold higher than the initial weight. Fibroblast viability and morphology showed a high cytocompatibility of the polyphenol-collagen mixtures. In conclusion, all these tests indicated an improved applicability of these mixtures for tissue wound healing.

Surgical Management of Post-repair Pulmonary Vein Stenosis

Obstructed pulmonary venous drainage - either in association with total anomalous pulmonary venous drainage, congenital stenosis, or post-repair stenosis - is associated with poor outcome. Post-repair stenosis typically involves fibrotic scar tissue extending from the site of anastomosis. "Sutureless" repair techniques avoid direct left atrial-pulmonary vein suture lines by instead reconstituting atrial tissue to posterior pericardium. Hence, the repair leaves widely decompressed pulmonary veins in the posterior mediastinum draining directly into the left atrium as a 'controlled bleed.' In our experience, late outcomes are significantly more favorable with sutureless repair techniques versus conventional pulmonary vein surgery. Therefore, after these encouraging results, we have now extended the application of this repair strategy to all scenarios of pulmonary vein surgery, including primary repair of unobstructed total anomalous pulmonary venous connection. The sutureless repair is versatile and facile. In particular, complex geometry of multiple decompressed veins can be easily accommodated by wide left atrial-pericardial suture lines. Common pitfalls can be avoided by mobilizing and protecting the phrenic pedicle and preserving the integrity of the areolar connective tissue and pleuro-parietal membrane. Overall, sutureless repair of anomalous or stenotic pulmonary veins appears safe and effective. Furthermore, in patients known to be at high risk of recurrent stenosis, sutureless techniques appear to offer improved freedom from recurrent stenosis.

Comparative expression pattern analysis of the highly conserved chemokines SDF1 and CXCL14 during amniote embryonic development

Chemokines are secreted proteins with essential roles in leukocyte trafficking and cell migration during embryogenesis. CXCL14 displays a degree of evolutionary conservation unmatched by any other chemokine except for SDF1(CXCL12). However, its role during embryogenesis has not been studied. Here we describe the expression pattern of mouse and chicken CXCL14 during embryogenesis and compare it with that of SDF1. CXCL14 is widely expressed in embryonic ectoderm and shows a restricted and dynamic expression pattern in paraxial mesoderm, mesonephros, neural tube, and limbs. During limb development, CXCL14 marks a unique connective tissue subset that surrounds developing tendons. Comparison of CXCL14 and SDF1 reveals mostly non-overlapping or complementary expression patterns, suggesting an interactive regulation of developmental processes by these two chemokines. Our study identifies CXCL14 as a novel marker of tendon connective tissue and provides a conceptual framework for the coordinated action of two highly conserved chemokines in embryonic development.

Micronutrient synergy—a new tool in effective control of metastasis and other key mechanisms of cancer

Consumption of a plant-based diet has been associated with prevention of the development and progression of cancer. We have developed strategies to inhibit cancer development and its spread by targeting common mechanisms used by all types of cancer cells that decrease stability and integrity of connective tissue. Strengthening of collagen and connective tissue can be achieved naturally through the synergistic effects of selected nutrients, such as lysine, proline, ascorbic acid and green tea extract (NM). This micronutrient mixture has exhibited a potent anticancer activity in vivo and in vitro in a few dozen cancer cell lines. Its anti-cancer effects include inhibition of metastasis, tumor growth, matrix metalloproteinase (MMP) secretion, invasion, angiogenesis, and cell growth as well as induction of apoptosis. Many cancers are often diagnosed at later stages, when metastasis has occurred, which standard treatment has been unable to control. Our studies on NM effects on hepatic and pulmonary metastasis demonstrated profound, significant suppression of metastasis in a murine model. Evaluation of effects of NM on xenografts in murine models demonstrated significant reduction in tumor size and tumor burden in all human cancer cell lines tested. In vitro studies demonstrated that NM was very effective in inhibition of cell proliferation (by MTT assay), MMP secretion (by gelatinase zymography), cell invasion (through Matrigel), cell migration (by scratch test), induction of apoptosis (by live green caspase) and induction of pro-apoptotic genes in many diverse cancer cell lines. Furthermore, in vivo and in vitro studies of effects of individual micronutrients compared to their specific combination demonstrated synergistic effects resulting in improved anticancer potency.

Comparative effects of EGCG, green tea and a nutrient mixture on the patterns of MMP-2 and MMP-9 expression in cancer cell lines

Type IV collagenase matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, have been found to promote invasion and metastasis of malignant tumors. Extracellular matrix (ECM) degradation by MMPs and increased expression of MMPs in cancer cells and tumor microvascular endothelial cells make MMPs an attractive target for cancer. Focused on a common pathomechanism of cancer growth and invasion, the disintegration of connective tissue, we used natural approaches to increase the integrity and strength of connective tissues. Utilizing the principle of nutrition synergy, we developed a novel micronutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract. This study evaluates the potency of the components EGCG and green tea extract independently compared to that of NM on modulation of patterns of MMP-2 and MMP-9 expression in four cancer cell lines expressing MMP-2, MMP-9 or both. Human fibrosarcoma (HT-1080), hepatocellular carcinoma (SK-Hep-1), glioblastoma (T-98G), uterine leiomyosarcoma (SK-UT-1) cell lines were obtained from ATCC and grown in minimum essential medium (MEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with agents dissolved in media and tested at concentrations indicated in triplicate at each dose. Cells were also treated with PMA 100 ng/ml to study enhanced expression of MMP-9. MMP expression was assessed by gelatinase zymography. Fibrosarcoma and hepatocellular carcinoma cells expressed both MMP-2 and MMP-9. Glioblastoma cells expressed MMP-2 and PMA treatment induced MMP-9 expression. Uterine leimyosarcoma cells expressed no MMPs but PMA induced MMP-9. NM was the most potent dose-dependent inhibitor of MMPs, followed by green tea extract and EGCG. In conclusion, these results suggest the enhanced efficacy of nutrients working in synergy to modulate complex pathways such as MMP expression.

Stem cells for tendon tissue engineering and regeneration

Importance of the field: Tendon injuries are common especially in sports activities, but tendon is a unique connective tissue with poor self-repair capability. With advances in stem cell biology, tissue engineering is becoming increasingly powerful for tissue regeneration. Stem cells with capacity of multipotency and self-renewal are an ideal cell source for tissue engineering.Areas covered in this review: This review focus on discussing the potential strategies including inductive growth factors, bio-scaffolds, mechanical stimulation, genetic modification and co-culture techniques to direct tendon-lineage differentiation of stem cells for complete tendon regeneration. Attempting to use embryonic stem cells as seed cells for tendon tissue engineering have achieved encouraging results. The combination of chemical and physical signals in stem cell microenvironment could be regulated to induce differentiation of the embryonic stem cells into tendon.What the reader will gain: We summarize fundamental questions, as well as future directions in tendon biology and tissue engineering.Take home message: Multifaceted technologies are increasingly required to control stem cell differentiation, to develop novel stem cell-based therapy, and, ultimately, to achieve more effective repair or regeneration of injured tendons.

Stimulation of the Extracellular Matrix Production in Dermal Fibroblasts by Velvet Antler Extract

Fibroblasts produce many components of the extracellular matrix (ECM) and so they contribute to the maintenance of connective tissue integrity. The aim of this study is to evaluate the effect of velvet antler extract (VAE) on the ECM production of dermal fibroblasts cultured in vitro. Primary cultured human dermal fibroblasts were treated with VAE, and then the ECM production was determined by RT-PCR, ELISA and Western blot analysis. Furthermore, the change of gene expression according to VAE treatment was evaluated by cDNA microarray. VAE accelerated the growth of fibroblasts in a dose-dependent manner. VAE increased the production of several ECM components, including type 1 collagen, fibronectin and elastin. In line with these results, the phosphorylations of p42/44 ERK and p38 mitogen-activated protein kinase were markedly increased by VAE, suggesting that the enhancement of ECM production may be linked to the activation of intracellular signaling cascades. VAE also significantly increased cell migration on an in vitro scratch wound test. In cDNA microarray, many genes related with connective tissue integrity were identified to be up-regulated by VAE. These results suggest that VAE has a potential to stimulate ECM production, and VAE may be applicable for maintaining the skin's texture.

Differential expression of the LOX family genes in human colorectal adenocarcinomas

Lysyl oxidase (LOX) is an amine oxidase that catalyzes the cross-linking of collage or elastin in the extracellular matrix, regulating the tensile strength and structural integrity of connective tissues. Recently, four paralogues (LOXL, LOXL2, LOXL3 and LOXL4) of LOX have been identified in humans, each containing the functional domains required for the amine oxidase activity toward collagen and elastin. Paradoxical roles of the LOX family members have been reported in various neoplastic tissues as tumor suppressors or promoters depending on tumor status and type. To address expression of the LOX family genes in colorectal adenocarcinomas, we performed real-time PCR analysis with matched tumor/normal tissue specimens from 104 patients. The expression of the LOX family genes was not statistically associated with tumor location, stage, growth type, or differentiation status. However, upregulation of LOX, LOXL2 and LOXL4 was significantly correlated with absence of lymphovascular invasion (P=0.012, 0.014 and 0.005, respectively), suggesting that the oxygen tension in or around the tumors may be an important regulator for the differential expression of LOX, LOXL2 and LOXL4 in colorectal cancer. Additionally, expression of LOX, but not the other LOX family genes, was significantly upregulated in patients with a diffuse cytoplasmic expression pattern of CEA, indicating that LOX upregulation may be associated with increased invasiveness and metastatic potential in colorectal cancer.

Formation of bone-like mineralized matrix by periodontal ligament cells in vivo: a morphological study in rats

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.

New Roles for Copper Metabolism in Cell Proliferation, Signaling, and Disease

Over the past 2 decades, the molecular mechanisms by which cells acquire, distribute, and utilize copper have been under intense investigation. Significant progress has been made in the identification of genes encoding copper homeostasis proteins and in fundamental aspects of their structure, function, and mechanisms of action in copper balance. A number of more comprehensive reviews of the field with respect to the genetics, structure, function, and physiology of copper metabolism have recently appeared elsewhere (1–4). Here, we review general mechanisms for eukaryotic copper metabolism at the cellular level in the context of recent discoveries in the field, identifying potential new functions for copper and copper metabolism proteins in cell signaling, gene expression, tumor cell metastasis, and resistance to anti-neoplastic drugs.

Prolidase Dependent Inhibition of Collagen Biosynthesis in Chinese Hamster Ovary Cells

Collagen is responsible for maintenance of connective tissue integrity, and through interaction with integrin receptors may participate in regulation of numerous physiological and pathological processes. An important role in collagen biosynthesis plays prolidase. It was previously found that nickel chloride inhibited prolidase activity in Chinese hamster ovary cells (CHO-C9). The cells lack any detectable ornithine aminotransferase and P5C synthase activities, and therefore require addition of free proline or glicyl-proline (converted to glycine and proline) for growth. We have found that Ni(II) contributed to decrease in collagen and hydroxyproline content in CHO cells incubated with Gly-Pro, whereas it had no effect on hydroxyproline content in the cells incubated with proline. Decrease in collagen content was not related to decrease in type I collagen mRNA level suggesting regulation of this process at post-transcriptional level. However decrease in expression of Sos and phosphorylated MAP-kinases were found in the cells growing in the presence of Gly-Pro and Ni(II). Decrease in the expression of these proteins was not related to inhibition of signalling induced by growth factors, since no changes were observed in expression of AKT in CHO cells incubated with Ni(II). The results presented provide evidence for important role of prolidase in collagen biosynthesis.