Protein Phosphatase의 억제와 Protein Kinase C의 활성이 Collagen 합성 및 유전자 발현에 미치는 영향

권오종,김인산,박낭운,조준승 慶北大學校 醫科大學 1995 慶北醫大誌 Vol.36 No.2

목적 : 이 연구는 섬유아세포에서 collagen유전자 발현을 조절하는 기전을 밝히는 노력의 일환으로 세포내 단백질의 인산화에 중요한 역할을 하는 protein phosphatase의 길항제이며 non-phorbol ester tumor promoter인 okadaic acid(OKA)와 protein kinase C의 활성제인 phorbol myristate acetate(PMA)가 정상섬유아세포를 비롯한 수종의 서로 다른 특성의 세포에서 collagen합성 및 유전자 발현에 어떤 영향을 미치는가를 조사하였다. 재료 및 방법 : 각종 섬유아세포 즉 사람 폐섬유아세포인 HEL 299 세포, 골아세포인 MC3T3-El세포, 생쥐 섬유아세포인 NIH3T3 세포, 사람피부섬유아세포인 HSF30세포, keloid 섬유아세포를 비롯하여 마우스 정상 섬유아세포인 Balb/c 3T3 세포를 virus로 형질전환시킨 K-balt세포, SV-T2 세포 및 M-MSV-BALB 세포 등을 적당한 조건하에 배양하여 일정한 상태로 만들고 배양액에 protein Phosphatase의 억제제인 OKA와 protein kinase C의 활성제인 PMA를 일정한 조건으로 첨가하고서 ^3H-proline을 넣어서 총 단백질과 collagen에 편입되는 방사능의 정도를 측정하여 이들의 collagen 합성에 미치는 영향을 산출하였다. 또 이들의 영향이 유전자 수준에서 작용하는지를 조사하기 위하여 Chomczgynski와 Sacchi법에 의한 총 RNA의 수출, Thu등과 Feinberg 및 Vogelstein법에 따라 northern blot hybridization을 실시하고, Labarca 및 Paigen의 방법을 이용하여 DNA양을 측정하였다. 결과 : OKA와 PMA는 세포마다 정도의 차이는 있지만 collagen합성을 농도에 비례하여 억제되었으며, 그리고 OKA의 효과는 PMA에 의해 증대되었다. OKA의 collagen합성억제 효과는 반응 12시간 뒤에 나타났으며 PMA의 효과는 반응 3시간째에 나타났다. 이것은 OKA와 PMA의 collagen합성에 대한 작용기전의 다름을 시사한다. 또한 OKA와 PMA은 정상 섬유아세포의 α_1,(Ⅰ)procollagen mRNA치를 현저히 감소시켰으며 이것은 OKA와 PMA의 collagen합성 억제작용이 유전자 발현의 억제에 의한 것임을 뜻한다. Collagen 합성이 정상 보다 현저히 증가된 keloid세포에서도 OKA와 PMA는 모두 상대적 collagen합성을 감소시켰다. 바이러스로 형질전환시킨 세포에서는 OKA와 PMA에 대한 반응이 이질적으로 나타나 형질 전환과정에서 생긴 세포내의 변화가 OMA와 PMA의 collagen합성조절 기전에 영향을 주었음을 시사한다. 결론 : 이상의 결과를 종합하면 OMA와 PMA는 둘다 결합조직세포의 α_1,(Ⅰ)procollagen 유전자발현을 억제함으로써 collagen합성을 감소시켰으나 그 작용양상은 달라서 서로 다른 기전으로 작용한다 할 수 있다. 또한 형질전환세포에서 OKA와 PMA의 collagen합성에 대한 작용이 이질적으로 나타나 이들의 collagen합성에 대한 작용이 세포의 형질전환고정에서 역시 서로 다른 영향을 받는다고 할 수 있다. In this study, effects of two well known chemicals to affect intracellular signal transduction pathways, okadaic acid (OKA), a protein phosphatase inhibitor, and phorbol myristate acetate (PMA), a protein kinase C activator, on collagen synthesis and mRNA levels in various cell lines were evaulated. Both of OKA and PMA inhibited collagen synthesis in a dose-dependent manner in several fibro blasts and osteoblast. In human fibroblasts, a suboptimal dose of PMA potentiated the inhibitory effect of OKA on collagen synthesis. Time couse study, however, showed that OKA inhibited collagen synthesis at 12 h and PMA at 3 h after treatment indicating that OKA and PMA may affect colla gen sythesis by different pathways. Northern blot anaysis showed that both of OKA and PMA decreased α_1[Ⅰ]procollagen mRNA levels in human fibroblasts and in this effect, they did not require a new protein synthesis. OKA and PMA also decreased collagen sythesis in several keloid fibroblasts of which collagen synthetic acitivities were elevated. In virally transformed fibroblasts, however, OKA and PMA showed quite different effects on collagen synthesis suggesting that intracellualr changes in the processes of viral transformation might effect the regulatory mechanisms of collagen synthesis and thus affect collagen synthesis in responses to OKA and PMA. Taken together, both of OKA and PMA inhibited collagen synthesis in several different cell lines but their actions were different mechanisms.

Keloid 세포의 Collagen 합성에 대한 Phorbol ester, Okadaic acid 및 Steroid의 작용

김원태,김인산,박낭운,손건영,조준승 慶北大學校 醫科大學 1995 慶北醫大誌 Vol.36 No.1

목적 : 이 연구는 Keloid 병변부위와 그 인접부위 그리고 정상부위의 세포에서의 collagen 합성에 대한 phorbol myristate acetate (PMA), okadaic acid (OKA) 및 hydrocortisone (HC)의 영향을 조사·비교 한 것이다. 재료 및 방법 : Keloid 병변부위와 그 인접부위 및 정상피부조직의 작은 절편을 배양하여 섬유아세포가 자라나와 밀생상태로 된후 trypsin 처리로써 섬유아세포만을 분리·계대 배양하였으며, 이 세포배양액에 PMA(100ng/㎖), OKA(10ng/㎖) 또는 HC(1.5μM)틀 부여, 24시간 배양해서 Peterkofsky 등의 방법에 따라 총단백질 및 collagen 합성능을 측정하여 대조군의 그것과 비교하였다. 결과 : Keloid 병변부위 및 그 인접부위의 세포는 정상부위의 세포보다 collagen 합성능이 높았다. 그 중에도 인접부위에서 가장 높았다. 그리고 PMA, OKA 및 HC는 다같이 비슷하게 또 부위에 관계없이 총단백질에 대한 collagen 합성비율을 억제하였다. 이들 약제중 PMA는 주로 noncollagen protein(NCP)을 많이 증가시켰고, OKA는 collagen 합성을 감소시켰다. 결론 : Keloid 병변부위와 그 인접부위의 세포에서 collagen 합성능이 높았다. 그리고 인접부위에선 더욱 PMA, OKA 및 HC에 의해 keloid 병변부위와 정상부위와는 달리 섬유아세포에서의 collagen 합성이 억제되었다. Keloid is a disease characterized by excessive deposition of various connective tissue components, such as collagen. In this study, three types of cells originate from keloid, keloid-adjacent and normal site of keloid patients were used to evaluate collagen synthesis by phorbol myristate acetate (PMA), okadaic acid (OKA) and hydrocortisone (HC). Average collagen synthetic rate by fibroblasts derived form keloid and keloid-adjacent cells was higher than that of normal skin fibroblasts. Keloid-adjacent cell has the most active collagen synthetic rate. PMA, OKA and HC inhibit % collagen synthesis in all three types of cells. In terms of noncollagen protein and collagen synthesis, PMA mainly increased noncollagen protein synthesis on the contrary OKA usually inhibit collagen synthesis in three types of cells. PMA, OKA and HC also inhibit % collagen synthesis in keloid and normal cells derived from the same patient similarity. Also there was no difference % collagen synthesis between keloid cells and keloid-adjacent cells derived from the same patient by three above mensioned reagents. Taken together, collagen synthetic rate was higher in keloid cells, when compared to the normal fibroblasts. In addition, there was no difference between keloid and normal cells in inhibitory effect of PMA, OKA and HC on collagen synthesis.

Keloid세포의 Collagen합성에 관한 연구

박혜경,박낭운,이병헌,최제용,김인산,조준승 慶北大學校 醫科大學 1995 慶北醫大誌 Vol.36 No.2

목적 :이 연구의 목적은 keloid가 점점 그 주위조직으로 침범하는 성질이 있는 것에 착안하여 keloid 중심부위와 그 인접부위 그리고 정상부위 조직에서의 collagen 합성능을 측정하여 상호비교 해보는데 있다. 재료 및 방법 : 7예의 keloid 섬유아세포와 3예의 keloid 인접부위 섬유아세포 그리고 5예의 정상부위 섬유아세포를 배양하여 Peterkofsky씨등의 방법^13)에 따라 collagen 합성능을 측정하였다. 해체와 배양조건에 따른 차이를 배제하기 위하여 한 환자로부터 동시에 각 부위의 조직을 동시에 얻어 동시에 섬유아세포를 배양하였다. 결과 : Collagen 합성능의 평균값은 정상조직보다 keloid 부위에서 훨씬 높았으며 또한 keloid 부위보다 그 인접부위에서 더욱 높았다. 결론 : 섬유아세포에서의 collagen 합성능은 정상부위, keloid 부위 및 그 인접부위 순으로 더욱 높았다. 이것은 keloid 환부주위에 국소적으로 작용하는 인자들이 존재하여 이들에 의해 keloid 주위조직이 자극을 받아 collagen 합성능이 증가하게 됨을 시사한다. Keloids are predominantly fibrous skin tumors that take the form of firm, variably pruritic or tender growths near the site of an injury. Histopathologically they are ricd in various connetive tissue components, such as collagen. In this study, the collagen synthesis by fibroblasts derived from keloid, keloid-adjacent area and normal skin was analyzed. Six keloid fibroblast cell lines out of 7 demonstrated increased collagen synthesis and one showed collagen synthetic rate that was within the control limits. One keloid fibroblast cell and one normal fibroblast cell were cultured from a patient, and when their collagen synthetic rates were compared with each other it resulted in that keloid cells showed a higer synthetic rate than normal cells. Three keloid-adjacent fibrolast cell lines showed markedly incerased collagen synthesis than their corresponding keloid cell lines which were derived from the same patients. Taken together, these results suggest that in keloid lesion, there might be local factors which stimulate surrounding fibroblasts to synthesize excessive collagen.

Effects of human collagen α-1 type I-derived proteins on collagen synthesis and elastin production in human dermal fibroblasts

( Su Jin Hwang ),( Su Hwan Kim ),( Woo-young Seo ),( Yelin Jeong ),( Min Cheol Shin ),( Dongryeol Ryu ),( Sang Bae Lee ),( Young Jin Choi ),( Kyeongjin Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2021 BMB Reports Vol.54 No.6

Collagen type I is the most abundant form of collagen in human tissues, and is composed of two identical α-1 type I chains and an α-2 type I chain organized in a triple helical structure. A previous study has shown that human collagen α-2 type I (hCOL1A2) promotes collagen synthesis, wound healing, and elastin production in normal human dermal fibroblasts (HDFs). However, the biological effects of human collagen α-1 type I (hCOL1A1) on various skin properties have not been investigated. Here, we isolate and identify the hCOL1A1-collagen effective domain (CED) which promotes collagen type I synthesis. Recombinant hCOL1A1-CED effectively induces cell proliferation and collagen biosynthesis in HDFs, as well as increased cell migration and elastin production. Based on these results, hCOL1A1-CED may be explored further for its potential use as a preventative agent against skin aging. [BMB Reports 2021; 54(6): 329-334]

<i>Pyropia yezoensis</i> peptide promotes collagen synthesis by activating the TGF-β/Smad signaling pathway in the human dermal fibroblast cell line Hs27

Kim, Cho-Rong,Kim, Young-Min,Lee, Min-Kyeong,Kim, In-Hye,Choi, Youn-Hee,Nam, Taek-Jeong UNKNOWN 2017 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.39 No.1

<P><I>Pyropia yezoensis</I> (<I>P. yezoensis</I>) is a marine algae that exhibits antioxidant, anti-inflammatory, antitumor and anti-aging activities. In this study, we investigated the effects of the <I>P. yezoensis</I> peptide, PYP1-5, on collagen synthesis in the human dermal fibroblast cell line Hs27. Skin aging is related to reduced collagen production and the activities of multiple enzymes, including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis, and tissue inhibitor of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the action of MMPs. While collagen synthesis is associated with a number of signaling pathways, we examined the increased collagen synthesis via the upregulation of the transforming growth factor-β (TGF-β)/Smad signaling pathway. Using MTS assay, we found that PYP1-5 did not affect cell viability. Moreover, we confirmed that PYP1-5 increased type 1 collagen expression using enzyme-linked immunosorbent assay (ELISA), western blot analysis and quantitative PCR. In addition, we identified changes in various enzymes, as well as the mechanisms behind the PYP1-5-induced collagen synthesis. PYP1-5 decreased the MMP-1 protein and mRNA levels, and increased the TIMP-1 and TIMP-2 protein and mRNA levels. In addition, PYP1-5 activated the TGF-β/Smad signaling pathway, which increased TGF-β1, p-Smad2 and p-Smad3 expression, while inhibiting Smad7, an inhibitor of the TGF-β/Smad pathway. Furthermore, PYP1-5 upregulated transcription factor specificity protein 1 (Sp1) expression, which is reportedly involved in type 1 collagen expression. These findings indicate that PYP1-5 activates the TGF-β/Smad signaling pathway, which subsequently induces collagen synthesis in Hs27 cells.</P>

Human collagen alpha-2 type I stimulates collagen synthesis, wound healing, and elastin production in normal human dermal fibroblasts (HDFs)

Su Jin Hwang,하근형,Woo-Young Seo,김충권,김경진,이상배 생화학분자생물학회 2020 BMB Reports Vol.53 No.10

Skin aging appears to be the result of overlapping intrinsic (including genetic and hormonal factors) and extrinsic (external environment including chronic light exposure, chemicals, and toxins) processes. These factors cause decreases in the synthesis of collagen type I and elastin in fibroblasts and increases in the melanin in melanocytes. Collagen Type I is the most abundant type of collagen and is a major structural protein in human body tissues. In previous studies, many products containing collagen derived from land and marine animals as well as other sources have been used for a wide range of purposes in cosmetics and food. However, to our knowledge, the effects of human collagenderived peptides on improvements in skin condition have not been investigated. Here we isolate and identify the domain of a human COL1A2-derived protein which promotes fibroblast cell proliferation and collagen type I synthesis. This human COL 1A2-derived peptide enhances wound healing and elastin production. Finally, the human collagen alpha-2 type I-derived peptide (SMM) ameliorates collagen type I synthesis, cell proliferation, cell migration, and elastin synthesis, supporting a significant anti-wrinkle effect. Collectively, these results demonstrate that human collagen alpha-2 type I-derived peptides is practically accessible in both cosmetics and food, with the goal of improving skin condition.

홍어 껍질 유래 콜라겐의 지방대사 조절을 통한 간 조직 지질축적 억제 효과

이현정(Hyun-Jung Lee),우민지(Minji Woo),송영옥(Yeong Ok Song),노정숙(Jeong Sook Noh) 한국식품영양과학회 2018 한국식품영양과학회지 Vol.47 No.3

홍어의 가공과정 중 발생하는 부산물인 껍질에서 추출된 콜라겐의 비만 쥐에서 지질대사에 미치는 영향에 대해 알아보고자 하였다. 랩틴 수용체의 유전적 결핍으로 사육 기간에 따라 비만 및 당뇨가 유발되는 동물모델인 db/db mice에 홍어 껍질 유래 콜라겐을 200 mg/kg bw/d로 8주 동안 경구투여하였고(콜라겐 투여군), 그 효과를 정상군(m/m mice) 및 비만대조군(db/db mice)과 비교하였다. 실험 8주의 비만쥐의 체중은 정상군보다 유의적으로 증가하였고, 콜라겐 투여군에서 비만 쥐에 비해 48.8% 감소하였다. 그러나 비만군과 콜라겐군 사이의 식이섭취량의 차이는 나타나지 않았다. 혈액의 중성지질과 총콜레스테롤의 농도는 비만군에서 정상군보다 증가하였는데, 콜라겐을 경구 투여한 결과 혈중지질의 농도는 각각 24.1% 및 29.3% 유의적으로 감소하였다. 또한, 간 조직의 중성지질과 총콜레스테롤의 함량을 측정한 결과도 혈액에서의 결과와 유사한 경향을 나타내었다. 콜라겐을 급여한 결과 비만대조군에 비해 중성지질과 총콜레스테롤의 농도는 각각 26.0% 및 8.5% 감소하였다. 간에서 홍어 껍질 유래 콜라겐의 지질합성 및 분해와 관련된 대사작용에 미치는 영향을 western blotting 방법을 통해 확인하였다. 지방합성 및 대사작용의 핵심 인자인 SREBP-1, SREBP-2, FAS, ACCα 등이 정상 쥐보다 비만 쥐의 간 조직에서 단백질 발현이 유의적으로 증가하였고, 콜라겐을 투여한 군에서 감소하였다. 또한, 에너지 생산계의 하나인 지방산 베타산화 인자인 PPARα와 ACOX-1은 반대로 비만 쥐의 간 조직에서의 발현이 정상 쥐보다 감소하였는데, 콜라겐 투여군에서 유의적으로 증가하였다. 따라서 본 연구의 결과로 살펴보았을 때, 효소적 가수분해를 통해 얻은 홍어 껍질 유래 콜라겐을 비만 쥐에 경구 투여하였을 때 체중증가를 억제하고, 혈액과 간의 지질 농도를 감소시켰다. 이는 콜라겐이 간에서의 지질합성 관련 인자의 억제 및 지방산 분해를 촉진하는 대사과정을 조절하여 비만에서 지질대사를 개선할 것으로 생각한다. Skate is a benthic animal found in the deep sea. Skate byproducts (skin, cartilage, and bone) are generated in large amounts and disposed of as waste during processing. Skate skin contains a high level of collagen, but few studies have investigated the health benefits of collagen beyond those on skin function. Therefore, this study was conducted to investigate the effects of collagen derived from skate skin on lipid metabolism in the liver using an obese animal model. To accomplish this, mice were divided into three experimental groups, Normal (m/m mice), Control (vehicle-treated db/db mice), and Collagen (200 mg/kg body weight collagen-treated db/db mice). Distilled water as the vehicle or collagen was orally administered to mice daily for eight weeks. Plasma and hepatic lipid levels were examined and the expression of hepatic proteins related to lipid metabolism were evaluated by western blotting analysis. Plasma and hepatic triglyceride and total cholesterol levels were significantly lower in the Collagen group (P<0.05), but were similar to those in the Normal group when compared to the Control. Additionally, expression of hepatic proteins associated with fatty acid synthesis (sterol regulatory element-binding proteins-1, fatty acid synthase, and acetyl-CoA carboxylase α) were decreased in the Collagen group, whereas phospho-activated protein kinase and beta-oxidation-related protein (peroxisome proliferator-activated receptor α and acetyl-CoA oxidase 1) were increased (P<0.05). Moreover, expression of proteins involved in cholesterol synthesis (sterol regulatory element-binding proteins-2) were decreased in collagen-treated mice, but those of the cytochrome P450 family 7 subfamily A member 1, which are involved in cholesterol export, increased (P<0.05). Our findings indicate that collagen derived from skate skin effectively suppressed hepatic lipid accumulation through down-regulation of fatty acid and cholesterol synthesis and up-regulation of beta-oxidation and cholesterol export.

L-Carnosine 및 트롬빈에 의한 인체 피부 섬유 모세포의 증식과 교원질 합성의 촉진효과

임풍,최동진,김영진 大韓成形外科學會 1995 Archives of Plastic Surgery Vol.22 No.5

It has been well known that the proliferation of fibroblasts plays a crucial role in wound healing process. In order to examine the effect of L-carnosine (β-alanyl-L-histidine) and thrombin on the proliferation and collagen synthesis of fibroblasts, DNA and collagen synthesis were studied in cultured human fibroblasts. The incorporation rates of [³H]-thymidine and [³H]-proline were measured at 2 hours or 5 hours after administration of L-carnosine or thrombin into the human skin fibroblasts cultures. The results were as follows; 1) The effective dose of L-carnosine was 1×10-²% in the incorporation rates of [³H]-thimidine into the cultured human skin fibroblasts. 2) The effective dose of thrombin was 1 unit in the incorporation rates of [³H]-thimidine into the cultured human skin fibroblasts. 3) The administration of L-carnosine (1×10-²%) or thrombin (1 unit) greatly increased DNA synthesis of the cultured human skin fibroblasts, whereas no significant increase of the DNA synthesis of the cultured human skin fibroblasts was shown in β-alanine-treated or L-histidine-treated group. 4) The administration of L-carnosine(1×10-²%) profundly increased collagen synthesis of the cultured human skin fibroblasts at 2 or 5 hours after the treatment. However, β-alanine, L-histidine or thrombin treatment slightly increaed the collagen synthesis of the cultured human skin fibroblasts at 2 or 5 hours after the treatment. These results demonstrate that the thrombin might enhance the proliferation of the cultured human skin fibroblasts, however, the L-carnosine might accelerate both proliferation and collagen synthesis.

Wound Healing Effect of Collagen-hyaluronic Acid Implanted in Partially Injured Anterior Cruciate Ligament of Dog

Seo, Young-Kwon,Park, Jung-Keug,Song, Kye-Yong,Kwon, Soon-Yong,Lee, Hwa-Sung 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.4

The purpose of this study was to evaluate the cell compatibility of collagen-hyaluronan (HA) substrate in vitro by assessing ACL cell cultures. Additionally, the use of collagen-HA substrate as a dressing material for partial ACL defects as well as its effect on collagen synthesis and angiogenesis were evaluated in vivo. The initial attachment and proliferation of dog ACL cells on silk matrix covered with collagen-HA substrate (SMCH) was greater than that observed on silk matrix alone. Silk matrix and SMCHs were implanted as dressing materials into partial ACL defects located at the knees of dogs, and they were harvested six weeks after implantation. A histological evaluation of the collagen-HA substrates revealed the presence of monocytes and the absence of giant cells in all cases. MT staining of the SMCH-grafted group showed a higher level of granulation tissue formation consisting of fibroblasts and collagen fibers compared to the silk matrix-grafted group. In addition, CD31 staining revealed that the SMCH-grafted area showed more blood vessel formation than the silk matrix-grafted area. These results suggest that the collagen-HA substrate was cell-compatible in vitro and enhanced collagen synthesis and new blood vessel formation in vivo.

Inhibitory Effects of Collagen Coated Coffee Bean Intake on Skin Aging

이인아,하미애,신용욱 인간식물환경학회 2019 인간식물환경학회지 Vol.22 No.1

To evaluate the protective effect of collagen peptide-coated coffee extract on skin aging, cell viability was measured with a MTT assay using cultured CCD-986sk fibroblasts, and its effect on wrinkles in the skin of hairless mice induced by UVB-irradiation was examined. In addition, its effect on procollagen synthesis and anti-oxidative, and its inhibitory activity against collagenase, elastase, tyrosinase and MMP-1 were analysed. After the 30-minute topical treatment, the animals were exposed to UVB irradiation (60-100 mJ/cm2) for 4 weeks and its intensity increased during the period. Under the experimental conditions set in this study, the skin thickness of hairless mice significantly decreased (11.8-21.3%) compared to the control group. Based on these results, the prolonged oral intake of a collagen peptide mixture with coffee is expected to significantly increase the synthesis of procollagen in dermal fibroblasts, thereby contributing to the alleviation of wrinkling and lowered elasticity due to structural damage to the dermal layer caused by UV. The oral intake of collagen-coated coffee contributes to increasing collagen biosynthesis in a dose-dependent manner and alleviates the symptoms of thickened keratin caused by UV irradiation. However, it did not inhibit the enzymes involved in skin aging, whitening, wrinkle improvement, and antioxidation. Based on the these results, it can be concluded that the intake of collagen peptide-coated coffee extract can be utilized as an alternative material for the prevention or treatment of diseases associated with photoaging.