肝臓は代謝の中心器官であり、炭水化物、タンパク質、脂質、およびその他の物質を代謝することによって体の恒常性に関与しています。さらに、解毒と再生は肝臓の特徴的な機能です。これらの能力は、食物毒素The liver is the central organ of metabolism and is responsible for the homeostasis of the body by metabolizing carbohydrates, proteins, lipids, and other substances. Furthermore, detoxification and regeneration are characteristic functions of the liver. These abilities are thought to have been acquired during the long evolutionary process to protect animals from catastrophic damage to liver tissues caused by food toxins (有毒化学物質)によって引き起こされる肝臓組織への壊滅的な損傷から動物を保護するために、長い進化の過程で獲得されたと考えられていますtoxic chemicals) [1]。.

肝再生のメカニズムは長年にわたって研究されており、数多くの報告が発表されています。これらの結果を統合した優れたレビュー記事もありますThe mechanisms of liver regeneration have been studied for many years, and numerous reports have been published. There are also excellent review articles integrating those results [11,2,2,33,4,5,6]。げっ歯類肝臓の. The 70%肝部分切除術(PHx)は、肝再生の研究モデルとして広く使用されています。たとえば、麻酔をかけたラットの肝臓の約70%を外科的に切除すると、残りの肝臓は自発的かつ迅速に細胞分裂と増殖を開始し、元の臓器の体積と重量に再生し、この応答は自動的に終了します partial hepatic resection of rodent livers (PHx) has been widely used as a research model for liver regeneration. For example, when approximately 70% of the liver of an anesthetized rat is surgically removed, the residual liver spontaneously and rapidly initiates cell division and proliferation, regenerating to the volume and weight of the original organ, and this response is automatically terminated [1,2]。この動物モデルでは、細胞増殖の開始の唯一のシグナルは局所的な損傷と組織の喪失であり、これはウイルス感染または化学物質誘発性肝障害からの肝再生のモデルよりも単純な実験システムになります。しかし、. In this animal model, the only signals for the initiation of cell proliferation are local wounding and tissue loss, making this a simpler experimental system than models of liver regeneration from viral infection or chemical-induced liver injury. Nevertheless, in vivo experin vivo実験系は、1つの事象に多くの要因が関与しているため、PHx後の肝再生の詳細なメカニズムを調べることは本質的に困難です。mental systems have inherent difficulties in examining the detailed mechanisms of liver regeneration after PHx, because many factors are involved in a single event.

したがって、研究者らは、より単純なTherefore, researchers used a simpler in vitro primary cultured hepatocyte system to examine the actin vitro初代培養肝細胞システムを使用して、個々の肝細胞成長因子(および候補物質)とその細胞内シグナル伝達経路の作用を調べました。初代培養肝細胞は、生体内と同等の代謝活性を保持し、多くの種類の受容体を発現しており、それぞれがアゴニストに応答することが知られていますons of individual hepatocyte growth factors (and candidate substances) and their intracellular signaling pathways. Primary cultured hepatocytes retain metabolic activity comparable to that in vivo and express many kinds of receptors, each of which is known to be responsive to agonists [3,7]。また、肝組織を構成する実質細胞. It is also known that, among the parenchymal (肝細胞)と非実質細胞(クプファー、ピット、星状、内皮など)の細胞のうち、肝細胞が最も早い段階で増殖を開始することも知られています。i.e., hepatocytes) and nonparenchymal (i.e., Kupffer, pit, stellate, endothelial, etc.) cells that constitute liver tissues, the hepatocytes initiate proliferation at the earliest stage.

2. 肝細胞増殖調節因子の分類と特徴Classification and Characteristics of Hepatocyte Growth Regulators

マイトジェンは、EGF、TGF-α、HGF、血小板由来増殖因子(PDGF)、IGF-Iなど、それ自体で肝細胞の増殖を促進する因子として定義することができる。コマイトジェンは、それ自体では肝細胞の増殖を促進しませんが、マイトジェンと組み合わせて使用 すると、コマイトジェンはマイトジェンの活性を増強し、これらにはアドレナリン作動性αおよびβアゴニストおよびグルカゴンが含まれます。さらに、TNF-α、IL-1β、プロスタグランジン(PG)Eなどの多くの間接マイトジェンがあります。

Mitogens can be defined as factors that promote the proliferation of hepatocytes by themselves, such as EGF, TGF-α, HGF, platelet-derived growth factor (PDGF), and IGF-I. Co-mitogens, by themselves, do not promote hepatocyte proliferation, but, when used in combination with mitogens, the co-mitogens enhance the activity of the mitogens, and these include adrenergic α- and β-agonists and glucagon. In addition, there are many indirect mitogens, such as TNF-α, IL-1β, prostaglandin (PG) E

₂、5-HT、およびGHは、自己分泌因子を分泌することによって肝細胞の増殖を間接的に促進する。対照的に、形質転換成長因子-β1(TGF-β1)やグルココルチコイドなどのいくつかの因子は、マイトジェン誘導肝細胞の増殖を強力に抑制します(すなわち、それらは抑制因子です)。

, 5-HT, and GH, which indirectly promote hepatocyte proliferation by secreting autocrine factors. In contrast, some factors, such as transforming growth factor-β1 (TGF-β1) and glucocorticoids, strongly suppress mitogen-induced hepatocyte proliferation (i.e., they are inhibitory factors).

2.1 マイトジェンMitogens

2.1.1 直接マイトジェンDirect Mitogens

ティッカー

EGF

EGFは分子量約 is a protein with a molecular weight of approximately 6kDaのタンパク質で、3つのジスルフィド結合を持っています。EGFはEGF受容体 kDa, and it has three disulfide bonds. EGF binds to the EGF receptor (EGFR)に結合し、受容体チロシンキナーゼ(RTK)をリン酸化することによって細胞内シグナル伝達経路を活性化します。EGF受容体は、PHxの30〜60分後にin vivoでリン酸化されます and activates intracellular signaling pathways by phosphorylating the receptor tyrosine kinase (RTK); the EGF receptor is phosphorylated in vivo 30–60 min after PHx [3]。. 肝細胞に対するThe proliferative effects of EGFの増殖効果は、培養で調べられています。その結果、EGF単独ではDNA合成と肝細胞の増殖が促進されることが示されています。RTK、細胞外シグナル調節キナーゼ(ERK)、およびラパマイシンの哺乳類標的 on hepatocytes have been examined in culture. The results have shown that EGF alone promotes DNA synthesis and the proliferation of hepatocytes. It has been suggested that RTK, extracellular signal-regulated kinase (ERK), and the mammalian target of rapamycin (mTOR)がシグナル伝達に関与していることが示唆されています are involved in the signaling [13]。ノルアドレナリンとアドレナリン作動性. Noradrenaline and an adrenergic β₂受容体アゴニストは、それ自体では肝細胞の増殖を促進しないが、これらの薬剤は receptor agonist, on their own, do not promote hepatocyte proliferation, but these agents enhance the growth-promoting effects of EGFの増殖促進効果を高める。したがって、アドレナリン作動性. Thus, there is crosstalk between the adrenergic β間にクロストークがあります₂受容体媒介シグナル伝達系および receptor-mediated signaling system and the EGF受容体媒介シグナル伝達系 receptor-mediated signaling system (図Figure 1)。.

TGF-α

TGF-α

TGF-αは分子量約 is a protein with a molecular weight of approximately 5.5 kDaのタンパク質で、EGFと高いアミノ酸相同性を持ち、, which has high amino acid homology with EGF and binds to EGFR/ErbB1に結合します。受容体二量体化後、TGF-αはRTKを活性化し、RTKは次に、細胞内シグナル伝達のためにアダプタータンパク質であるSmadを介してマイトジェン活性化タンパク質(MAP)キナーゼカスケードを活性化します. After receptor dimerization, TGF-α activates RTK, which, in turn, activates the mitogen-activated protein (MAP) kinase cascade via Smad, an adaptor protein, for intracellular signal transduction [35]。ケラチノサイトやさまざまな癌細胞によって産生されるサイトカインである. TGF-αは、培養中の肝細胞単独のDNA合成と増殖を直接促進します, a cytokine produced by keratinocytes and various cancer cells, directly promotes DNA synthesis and the proliferation of hepatocytes alone in culture [19]。また、いくつかのサイトカインに応答して肝細胞によって分泌される自己分泌因子でもあります. It is also an autocrine factor secreted by hepatocytes in response to several cytokines (以下を参照)。see below). 肝細胞に対するThe effects of TGF-αの効果は、培養において調査されています。その結果、TGF-α単独ではDNA合成と肝細胞の増殖が促進され、RTK、ホスファチジルイノシトール-3キナーゼ on hepatocytes have been investigated in culture. The results have shown that TGF-α alone promotes DNA synthesis and the proliferation of hepatocytes and that RTK, phosphatidylinositol-3 kinase (PI3K)、ERK、およびmTORがそのシグナル伝達に関与していることが示されています。さらに、アドレナリン作動性, ERK, and mTOR are involved in its signal transduction. In addition, adrenergic α₁受容体アゴニストは1 receptor agonists enhance the effects of TGF-αの効果を増強し、アドレナリン作動性, suggesting crosstalk with adrenergic αとのクロストークを示唆している₁受容体媒介性シグナル伝達経路 receptor-mediated signaling pathways (図Figure 1)。.

ティッカー

HGF

HGFは、 is a protein consisting of 728個のアミノ酸とヘテロ二量体構造からなるタンパク質で、重鎖は約60 kDa、軽鎖は約 amino acids and a heterodimeric structure, with a heavy chain of approximately 60 kDa and a light chain of 3.5 kDaです。肝臓の星細胞や内皮細胞が産生するサイトカインです。その受容体はcMetであり、細胞内ドメインのRTKを二量体化および活性化してHGFの作用を媒介します. It is a cytokine produced by stellate cells and endothelial cells in the liver. Its receptor is cMet, which dimerizes and activates the RTK in the intracellular domain to mediate the action of the HGF [36,37,38]。. 肝細胞に対するThe proliferative effect of HGFの増殖効果は、培養において調べられている。結果は、HGFのみがDNA合成と肝細胞の増殖を促進し、RTK、PI3K、ERK、およびmTORがそのシグナル伝達に関与していることを示しています on hepatocytes has been examined in culture. The results have shown that HGF alone promotes DNA synthesis and the proliferation of hepatocytes and that RTK, PI3K, ERK, and mTOR are involved in its signaling [20]。我々はまた、アドレナリン作動性. We have also found that both adrenergic α₁および and β₂受容体アゴニストは、クロストークによって receptor agonists potentiate the growth-promoting effects of HGFの成長促進効果を増強します by crosstalk (図Figure 1)。.

PDGF

PDGF belongs to the PDGF/VEGF family. Its molecular weight is approximately 30 kDa, and its A and B chains form homo- and hetero-dimeric structures (PDGF-AA, PDGF-BB, and PDGF-AB), which activate RTK for intracellular signal transduction [39]. It was first isolated from platelets, but it is also produced by macrophages, vascular endothelial cells, smooth muscle cells, and cancer cells. In addition to PDGF, the platelets also contain TGF-β1, HGF, and 5-HT, which are released during the platelet adhesion and aggregation associated with tissue injury [3]. The released PDGF is thought to trigger a series of responses including inflammation and macrophage, neutrophil, and fibroblast migration. We investigated the proliferative effects of PDGF on hepatocytes in culture. The results showed that PDGF-BB alone promotes DNA synthesis and proliferation of hepatocytes and that RTK, PI3K, ERK, and mTOR are involved in its signal transduction [21]. In addition, an adrenergic α₁ receptor agonist enhances the effects of the PDGF-BB, suggesting crosstalk with adrenergic α₁ receptor-mediated signaling pathways (Figure 1).

Insulin

Human insulin is a heterodimer consisting of an A chain of 21 amino acids and a B chain of 30 amino acids, joined by two disulfide bonds. It is produced by pancreatic B cells and can always act on the liver via the portal bloodstream. The receptor for insulin is a built-in tyrosine kinase, and its activation phosphorylates its intracellular substrate, insulin receptor substrate-1 (IRS-1). Subsequently, signals are transmitted to PI3K and protein kinase B, and glucose transporter-4 is translocated to the cell surface. It has also been reported that blood levels of insulin increase soon after PHx [40]. The proliferative effect of insulin on hepatocytes has been said to be a co-mitogen that enhances the action of direct mitogens such as EGF. Therefore, we investigated the effects of insulin in culture. Our results showed that insulin alone promotes DNA synthesis and hepatocyte proliferation and that RTK, PI3K, ERK, and mTOR are involved in its signaling [22,41]. In addition, an adrenergic α₁ receptor agonist potentiates the action of insulin, suggesting crosstalk with adrenergic α₁ receptor-mediated signaling pathways (Figure 1).

2.1.2. Co-Mitogens

Noradrenaline

Noradrenaline is a sympathetic neurotransmitter with a catecholamine structure. Noradrenaline receptors include adrenergic α and β types, each of which has subtypes. All the receptors for noradrenaline are G-protein-coupled ones. It has been reported that sympathetic hyperactivity due to post-PHx invasion increases the blood levels of noradrenaline in about 1 h [42]. It activates the duodenal Brunner’s gland and stimulates EGF production and HGF expression [43,44]. In primary cultured hepatocytes, noradrenaline alone does not promote the proliferation of hepatocytes, but, through crosstalk, it can enhance the hepatocyte proliferation-promoting effects of EGF [13], TGF-α [19], HGF [20], PDGF [21], and insulin [22] (Figure 1). In addition, in combination with phenylephrine (a selective adrenergic α₁-receptor agonist), it enhances the hepatocyte-proliferative effects of EGF, HGF, TGF-α, PDGF, and insulin. Metaproterenol (a selective β₂-receptor agonist) enhances the proliferative action of EGF, IGF-I, and HGF. These results show that noradrenaline exhibits unique regulatory mechanisms for these growth factors.

2.1.3. Indirect Mitogens

IL-1β

IL-1β was the first inflammatory cytokine among the ILs to be identified, and there are two types, IL-1α and IL-1β [45]. IL-1β is a protein consisting of 110–140 amino acids. IL-1 receptors are highly homologous to toll-like receptors and activate serine/threonine kinases via adaptor proteins for intracellular signal transduction. IL-1 is rarely detected in normal tissues and is produced and secreted by macrophages and other immune cells activated by infiltrating inflammation. It has proliferative effects on monocytes and granulocytes. The proliferative effects of IL-1β on hepatocytes have been examined in culture. The results have shown that IL-1β alone promotes DNA synthesis and the proliferation of hepatocytes and that the IL-1β effects are mediated via autocrine secretion of TGF-α from hepatocytes (Figure 2) [24]. IL-1α does not promote the proliferation of hepatocytes.

TNF-α

TNF-α is a protein consisting of 157 amino acids and is a potent proinflammatory cytokine similar to IL-1 and IL-6. TNF-α promotes the proliferation of hepatocytes via TNF-α type 1 receptors [46]. The major TNF-α-producing cells in peripheral tissues are macrophages (Kupffer cells in the liver), but it is also released from mast cells. It is involved in the pathogenesis of chronic inflammatory diseases such as Crohn’s disease and rheumatoid arthritis. We investigated the proliferative effects of TNF-α on hepatocytes in culture. The results showed that TNF-α alone promotes DNA synthesis and the proliferation of hepatocytes and that its effects are mediated via its autocrine secretion of TGF-α from the hepatocytes (Figure 2) [25].

PGE₂ and Prostacyclin (PGI₂)

Prostaglandins (PGs) are low-molecular-weight local hormones. The receptors for PGs include EP, IP, and TP types, all of which are G protein-coupled. PGs are synthesized under the influence of cyclooxygenase (COX) from arachidonic acid and excised from the plasma membrane by phospholipase A₂. The physiological process involves constitutive COX-1 expression, while the inflammatory process involves inducible COX-2 expression. Biosynthesized PG products exhibit a variety of physiological effects, but inactivation is relatively rapid. PGs act on cells of the immune system to enhance inflammatory responses (redness, fever, swelling, pain, and dysfunction) by promoting the release of IL-1 and TNF-α. PGI₂ is produced by vascular endothelial cells, regulates blood flow, and inhibits platelet aggregation. It has been reported that PGs stimulate hepatocyte DNA synthesis in culture [47]. In primary cultured hepatocytes, we investigated the intracellular signaling pathways of PGE₂ and PGI₂, which promote DNA synthesis and the proliferation of hepatocytes. As a result, we found that both PGE₂ and PGI₂ indirectly promote DNA synthesis and the proliferation of hepatocytes via the secretion of the autocrine factor TGF-α from the hepatocytes (Figure 2) [39,40,48].

5-HT

5-HT is a low-molecular-weight substance with receptor subtypes designated 5-HT₁ to 5-HT₇. 5-HT acts as a neurotransmitter in the central nervous system and as a messenger molecule in the periphery. For example, it is released from enterochromaffin (EC) cells in the small intestinal epithelium and acts as a local hormone. 5-HT is stored in platelets and released upon stimulation and is involved in blood coagulation by promoting platelet aggregate formation (hemostatic thrombus). 5-HT has been reported to be involved in the promotion of liver regeneration [49]. That is, thrombocytopenic mice have impaired liver regeneration, and the administration of 5-HT restores liver regeneration. However, since platelets also contain other mediators (PDGF, HGF, etc.) that promote hepatocyte proliferation, it is necessary to examine whether this is a direct or indirect action of 5-HT. When we examined the direct action of 5-HT on the proliferation of hepatocytes in culture, we found that 5-HT promotes the autocrine secretion of TGF-α from the hepatocytes via the 5-HT_2B/Gq/phosphoinositide-specific phospholipase C (PLC)/Ca²⁺ pathway. We then found that the secreted TGF-α directly promotes DNA synthesis and hepatocyte proliferation (Figure 2) [14,17,18].

GH

GH is a single-chain peptide consisting of 191 amino acids, which is secreted from the anterior pituitary gland. GH induces functional changes in the metabolic capacities of various organs, such as the growth and differentiation of cells and tissues, bone mineralization, and the metabolism of carbohydrates, lipids, and proteins [28]. Its receptor is a Janus kinase 2 (JAK2)-related type. In vivo, GH is known to promote liver regeneration in PHx rats [50]. We examined the effects of GH on the proliferation of hepatocytes and intracellular signaling pathways in culture. GH promotes the autocrine secretion of IGF-I from the hepatocytes via the GH receptor/JAK2/PLC/Ca²⁺ pathway. We then found that the secreted IGF-I directly promotes DNA synthesis and hepatocyte proliferation (Figure 2) [15,16,23].