Procollagen Degradation Research Articles

Eisenia bicyclis Extract Repairs UVB-Induced Skin Photoaging In Vitro and In Vivo: Photoprotective Effects.

Chronic exposure to ultraviolet B (UVB) is a major cause of skin aging. The aim of the present study was to determine the photoprotective effect of a 30% ethanol extract of Eisenia bicyclis (Kjellman) Setchell (EEB) against UVB-induced skin aging. By treating human dermal fibroblasts (Hs68) with EEB after UVB irradiation, we found that EEB had a cytoprotective effect. EEB treatment significantly decreased UVB-induced matrix metalloproteinase-1 (MMP-1) production by suppressing the activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling and enhancing the protein expression of tissue inhibitors of metalloproteinases (TIMPs). EEB was also found to recover the UVB-induced degradation of pro-collagen by upregulating Smad signaling. Moreover, EEB increased the mRNA expression of filaggrin, involucrin, and loricrin in UVB-irradiated human epidermal keratinocytes (HaCaT). EEB decreased UVB-induced reactive oxygen species (ROS) generation by upregulating glutathione peroxidase 1 (GPx1) and heme oxygenase-1 (HO-1) expression via nuclear factor erythroid-2-related factor 2 (Nrf2) activation in Hs68 cells. In a UVB-induced HR-1 hairless mouse model, the oral administration of EEB mitigated photoaging lesions including wrinkle formation, skin thickness, and skin dryness by downregulating MMP-1 production and upregulating the expression of pro-collagen type I alpha 1 chain (pro-COL1A1). Collectively, our findings revealed that EEB prevents UVB-induced skin damage by regulating MMP-1 and pro-collagen type I production through MAPK/AP-1 and Smad pathways.

The Antiaging Activity of Ergothioneine in UVA-Irradiated Human Dermal Fibroblasts via the Inhibition of the AP-1 Pathway and the Activation of Nrf2-Mediated Antioxidant Genes

UVA irradiation induced ROS-mediated photo damage to the human skin leading to coarseness, wrinkling, pigmentation, and cutaneous malignancies. We investigated the dermatoprotective efficacies of submicromolar concentrations of ergothioneine (EGT, 0.125-0.5 μM), which occurs naturally as a sulfur-containing amino acid, in the mechanisms in human skin fibroblast (HSF) cells. UVA-induced AP-1 (c-Fos and c-Jun) translocation was found to be inhibited by EGT treatments with the parallel inhibition of the collagenolytic matrix metalloproteinase- (MMP-) 1 activation and type I procollagen degradation. Moreover, EGT mitigated UVA-induced ROS generation. An increase in the amount of antioxidant genes (HO-1, NQO-1, and γ-GCLC) from EGT and were associated with upregulated Nrf2 expressions in a dose-dependent or time-dependent manner. We confirmed this from Nrf2 translocation and increased nuclear ARE promoter activity that underlie EGT dermatoprotective activities. Also, glutathione (GSH) levels (from γ-GCLC) were significantly increased. Moreover, we showed that mediated by ERK, JNK, and PKC, signaling cascades mediate Nrf2 translocation. We confirmed this phenomenon by the suppressed nuclear Nrf2 activation in cells that were treated with respective inhibitors (PD98059, SP600125, and GF109203X). However, antioxidant protein expressions were impaired in Nrf2 knockdown cells to confirm that ARE/Nrf2 pathways and the inhibition of AP-1 had significant roles in EGT-mediated protective effects. We can conclude that ergothioneine ameliorated UVA-induced skin aging and is a useful food supplement for skin care products.

Fermentation of Blackberry with L. plantarum JBMI F5 Enhance the Protection Effect on UVB-Mediated Photoaging in Human Foreskin Fibroblast and Hairless Mice through Regulation of MAPK/NF-κB Signaling.

Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.

Anti-photoaging effects of solvent-partitioned fractions from Portulaca oleracea L. on UVB-stressed human keratinocytes.

In this study, the anti-photoaging activity of solvent-fractionated extracts from Portulaca oleracea L. was investigated in ultraviolet B-stressed keratinocytes. The results showed that the treatment of the fractions suppressed the generation of intracellular reactive oxygen species (ROS) and activated mRNA and protein expression levels of superoxide dismutase1 (SOD-1) and heme oxygenase1 (HO-1) enzymes via Nrf-2 pathway. The tested fractions down-regulated the expression of matrix metalloproteinases (MMPs) and up-regulated type Ⅰ procollagen production. Among the tested samples, the 85% aq. MeOH fraction was the most effective in suppressing MMPs and type Ⅰ procollagen degradation. Two homoisoflavonoids, portulacanone A (PA) and portulacanon D (PD), were isolated from the 85% aq. MeOH fraction. PA and PD successfully inhibited the secretionof MMP-1 and increased the production of type Ⅰ procollagen in UVB-stressed keratinocytes. Therefore, solvent-partitioned factions from P. oleracea and its active components, PA and PD, were suggested to have beneficial effects against photoaging. PRACTICAL APPLICATIONS: Portulaca oleracea L. is an edible nutrient-rich herb with high amounts of omega-3 and omega-6 fatty acids, ascorbic acid, and dietary minerals. P. oleracea has also been used as a botanical medicine for its potential health benefits such as antioxidant, antibacterial, and antiinflammatory properties. The current study suggested that the extracts from P. oleracea were able to prevent human keratinocytes from UVB-induced stress and damage. Due to its protective abilities, it can be used as an additive in the cosmeceutical industry to produce novel skin protectants. In addition, due to its intracellular antioxidant and MMP inhibitory effects, it can be a valuable source for nutraceutical development. Further studies that characterize the isolated flavonoids may also yield bioactive molecules to be utilized in cosmeceutical and nutraceutical industries as lead compounds. Nevertheless, P. oleracea can serve as a health beneficial addition to topical applications and dietary supplements against UVB-stimulated oxidative stress in skin cells.

Potential prevention effects of Rubus occidentalis seed on UVB-induced MMP-1 production and procollagen degradation in CCD-986sk cells

UV exposure induces matrix metalloproteinases (MMPs) and extracellular matrix-degrading enzymes expression. We studied the protective effect of Rubus occidentalis seed against UVB-generated skin photoaging using human fibroblast cells (CCD-986sk). We used an ELISA kit to measure the supernatents of ...

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload

BackgroundPrevious work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. Methods and resultsIsolated LV fibroblasts taken from 4 and 12week ACF Sprague–Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. ConclusionIn a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague–Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

In Vitro and In Vivo Studies on Protective Action of N-Phenethyl Caffeamide against Photodamage of Skin.

In our previous study, N-phenethyl caffeamide (K36) was proved to act as an antioxidant and an antiphotoaging agent by inhibiting type I procollagen degradation and stimulating collagen synthesis in human skin fibroblasts. In the present study, in vitro and in vivo experiments were conducted to investigate the mechanism of action and the antiinflammatory and antiphotoaging activity of K36. K36 reduced UVB-induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS) expression by regulating IκB and p-IκB expression. K36 also inhibited the nuclear translocation of NF-κB. Furthermore, the inhibition of mitogen-activated protein (MAP) kinases by K36 was attributed to the downregulation of COX-2. Topically applying K36 led to efficient antiwrinkle formation and reduced UVB-induced erythema and thickness of epidermis in hairless mice. In addition, K36 penetrated into the skin of hairless mice. Our findings show that K36 has significant beneficial effects on antioxidant, antiinflammatory, and antiphotoaging activity and suggest that K36 can be developed as an antiaging agent for cosmetic and skin care products.

N-phenethyl caffeamide and photodamage: Protecting skin by inhibiting type I procollagen degradation and stimulating collagen synthesis

Skin is mainly damaged by genetic and environmental factors such as ultraviolet (UV) light and pollutants. UV light is a well-known factor that causes various types of skin damage and premature aging. Reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases that break down type I collagen. This study investigated the antioxidant and antiphotodamage activity and mechanisms of N-phenethyl caffeamide (K36) in human skin fibroblasts. The results indicated that K36 demonstrated strong 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, which dose-dependently reduced the production of UVB-induced intracellular ROS in human dermal fibroblasts. K36 prevented UVB-irradiation-induced type I collagen degradation by inhibiting the expression of matrix metalloproteins-1, -3, and -9 and the phosphorylation of mitogen-activated protein (MAP) kinases. Furthermore, K36 elevated collagen synthesis in skin fibroblasts by inhibiting UVB-induced Smad7 overexpression. K36 downregulated the expression of the transcription factor, activator protein-1 (AP-1). Our results indicated that K36 exhibited antioxidant properties and prevented skin collagen degradation caused by UV exposure and the stimulation of collagen synthesis, which suggests the potential use of K36 in preventing photodamage.

Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.

PDI-mediated ER retention and proteasomal degradation of procollagen I in corneal endothelial cells

Procollagen I in corneal endothelial cells (CECs) is intracellularly degraded immediately after its synthesis. In this study, we investigated the mechanism of intracellular degradation of procollagen I by determining the role of protein disulfide isomerase (PDI) in endoplasmic reticulum (ER) retention and further determined the degradation pathway of procollagen I in CECs. When association of PDI to monomeric proα chains or the trimeric procollagen I carboxyl propeptides (PICPs) was analyzed, immune complex precipitated with anti-PICP antibody contained more PDI than that precipitated with antibodies to monomeric chains. PICPs were completely colocalized with PDI. When CECs were transfected with PDI vector, procollagen I and the recombinant PDI were colocalized in the ER, whereas CECs transfected with PDI minus KDEL (the ER retrieval sequence) vector demonstrated that the two proteins were localized in the Golgi and were subsequently secreted into the medium. Ribostamycin (an inhibitor of the chaperone activity of PDI) blocked colocalization of PDI and procollagen I. Cells treated with chloroquine (lysosome inhibitor) did not alter the subcellular localization of procollagen I, because the inhibitor failed to induce the accumulation of procollagen I at Golgi. On the other hand, procollagen I was colocalized with ubiquitin in the cytoplasm, and proteasomal inhibitors further facilitated the colocalization of the two proteins and accumulation of ubiquitinated procollagen I ladders. These results suggest that association of PDI with procollagen I, whether monomeric or trimeric, leads to ER retention of procollagen I before intracellular degradation via the ubiquitin–proteasome pathway.

Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability

Extracellular matrix remodelling occurs during atherosclerosis dictating the structure of the plaque and thus the resistance to rupture. Monocytes and macrophages are believed to play a role in this remodelling. In the present study, filter-separated co-culture has been used to study the effect of monocytes on procollagen turnover by human vascular smooth muscle cells (VSMC). In this system, freshly isolated human peripheral blood monocytes inhibited procollagen secretion from VSMC without affecting either degradation of procollagen, or DNA synthesis by the VSMC. Insertion of a 12 kDa dialysis membrane between the two cell types and treatment with indomethacin showed that the inhibitory factor was of low molecular weight and was cyclooxygenase-dependent. Pre-incubation of each cell type with indomethacin demonstrated that monocyte, but not VSMC cyclooxygenase was required. Thus, the inhibitory effect on procollagen secretion was due, most likely, to monocyte prostaglandins. Neither inhibition of thromboxane synthetase, nor blocking IL-1 activity, reduced the inhibitory activity. Addition of prostaglandins PGE 1, PGE 2 and PGF 2 α to VSMC cultures caused a reduction in procollagen secretion which was equivalent to, but was not additive with, the maximal effect achieved by monocytes. Monocytes and macrophages are a major source of prostaglandins and these molecules are likely to play an important role in collagen turnover within lesions.

Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum.

Heat shock protein (HSP) 47 is a collagen-binding stress protein localized in the endoplasmic reticulum (ER). In addition to stress-inducibility through heat shock element-heat shock factor interaction, the expression of HSP47 under normal conditions always correlates with that of collagens in various cell types and tissues. Both HSP47 and types I and III collagens are also dramatically induced under pathophysiological conditions such as liver fibrosis. HSP47 transiently associates with procollagen in the ER and dissociates from it in the cis-Golgi compartment. Possible functions of HSP47 as a molecular chaperone specific for procollagen are discussed: prevention of nascent procollagen chains from forming aggregates, effect on the modification of procollagen, inhibition of intracellular degradation of procollagen, quality control mechanisms under stress conditions, and effect on the secretion from the ER to the Golgi compartment.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor-beta 1.

1. Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2. Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor-beta 1 (TGF-beta 1) for different time periods in the presence or absence of PTF. TGF-beta 1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3-6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9-12 h after the addition of TGF-beta 1. Co-incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell-associated expression of the corresponding protein. Conversely, PTF dose-dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3. Pulse-chase experiments employing L-[3H]-proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP-1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. Accordingly incubation with PTF increased the levels of 'activated MMP-1' in cell supernatants in both basal and stimulated conditions. 4. These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Prolyl hydroxylation regulates intracellular procollagen degradation in cultured rat cardiac fibroblasts

To determine the regulatory role of prolyl hydroxylation in intracellular cardiac procollagen turnover, we examined the effects of prolyl 4-hydroxylase inhibitors (alpha, alpha-dipyridil, 3,4-dihydroxybenzoic acid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascorbic acid on procollagen metabolism by cultured, neonatal rat cardiac fibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a significant reduction in alpha 1(I) and alpha 1(III) mRNA levels. The fraction of newly synthesized procollagens degraded intracellularly was also substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P < 0.05). These findings were associated with increased intracellular accumulation of Type I procollagen, enhanced secretion of "underhydroxylated" pro alpha 1 (I) polypeptide into the cell culture medium, and decreased extracellular Type I collagen deposition. Similar results were obtained by treating cells with alpha, alpha-dipyridil (300 microns), and 3,4-dihydroxybenzoic acid ethyl ester (400 microM) in the presence of ascorbate. A major portion of the enhanced degradation of newly synthesized procollagens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 microM). Inhibition of procollagen secretion by colchicine (0.5 micrograms/ml) enhanced the diversion to, and subsequent intracellular degradation of underhydroxylated procollagens in cardiac fibroblast lysosomes. We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accumulation and intralysosomal degradation of newly synthesized cardiac procollagen polypeptides. These observations suggest that procollagen prolyl hydroxylation may be important in the regulation of collagen accumulation by cardiac interstitial cells during fibrotic processes in vivo

Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein

Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.

Inhibition of Procollagen I Degradation by Colligin: A Collagen-Binding Serpin

Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment.

A disorder of collagen biosynthesis in patients with cerebral artery aneurysm

12 patients with subarachnoid hemorraghe due to rupture of a cerebral aneurysm were examined clinically for symptoms and signs of a connective tissue disorder and biochemically for details of the biosynthesis of collagen. No uniform clinical pattern of any connective tissue disorder was seen in these patients, although selected signs were observed. Skin fibroblast cultures were then established. The rate of procollagen production in two cell lines was reduced by 40% and 50%, respectively, and the intracellular accumulation of hydroxyl[ 14C]proline (as a percentage of total hydroxy[ 14C]proline) was increased by 70% in each relative to eight control cell lines. No difference was found in the degree of intracellular degradation of procollagen. After pulse-labelling, however, the radioactive procollagen was secreted into the medium in 1 h in the control cells, but required at least 3 h in the two aneursym patient cell lines. The results, thus, suggest that delayed secretion of procollagen rather than increased intracellular degradation led to the reduction in the rate of procollagen synthesis in these two fibroblast lines from patients with cerebral artery aneurysm.

Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.

Minocycline-Treatment of Diabetic Rats Normalizes Skin Collagen Production and Mass: Possible Causative Mechanisms

Daily minocycline-treatment of streptozotocin-induced diabetic rats not only prevented a diabetes-caused atrophy of skin collagen mass (IO-mos old rats), but also normalized skin collagen mass to match that of growing (ca.1 %/d) non-diabetic controls (4- and 5-mos old rats).The causative mechanism by which minocycline-treatment normalizes skin collagen mass must, in part, be related to a general anabolic effect on growth (body weight) because the effect on skin collagen mass correlates strongly to that on body weight.Consequently, a minocycline-stimulated increase of a systemic factor (such as insulin-like growth factor) is not unlikely.The anabolic effect of minocycline-treatment of diabetic rats is also expressed as a normalized cellular ribosome mass (an index of total protein synthetic capacity) and a normalized absolute rate of collagen production.(Calculation of an absolute rate was justified by an apparent maximum saturation of the prolyl-tRNA pool(s) of skin, maximum saturation obtained by the pool-flooding approach.)The normalized skin ribosome amount does not, however, explain a selective effect of minocylcine-treatment on collagen production as opposed to that for non-collagen protein, this selective effect measured as relative collagen production.To explain such selectivity, the inhibition of diabetes-induced excess skin collagenase activity seems unlikely.(This inference is based on results from a preliminary study indicating that recently [2 hs] synthesized collagen is not degraded by the excess collagenase in skin of diabetic rats.) Thus, the principal collagen fraction acted on by pathologically excess collagenase might be collagen at a later stage (> 2 h after synthesis) in its life cycle.(Another possibility for the selective effect of minocycline on collagen production, as yet untested, is reduced intracellular pro collagen degradation.) Overall, this is the first study aimed at discerning the mechanism(s) by which minocyclinetreatment enhances the rate of collagen production in tissues of a diabetic rat.For future studies, the extent to which the positive effect on growth, ribosome mass, and rate of collagen production contributes to the change of collagen mass must, along with the known minocycline-inhibition of collagenase activity, be quantified.Such quantification is a prerequisite for evaluating the chemotherapeutic efficacy of minocycline-treatment on collagen-degradative diseases.