Keratinocyte of spinosum layer differentiates into keratinocyte of granulosum layer in interfollicular epidermis

Spinous keratinocytes form 4-8 layers of cells in the stratum spinosum (spinous layer) of the interfollicular epidermis (reviewed in Fuchs 1990). While spinous layer keratinocytes are considered to be postmitotic (reviewed in Fuchs 1990), they can still be positive for some of the proliferation markers seen in transit amplifying cells, such as MKI67 and histone H3 phosphorylated on serine residue S11. Currently, generic markers of cell proliferation are not annotated in Reactome. The percentage of spinous layer keratinocytes positive for mitotic markers decreases with age and differs between different skin areas (e.g. plantar skin vs. trunk skin) (Nöske et al. 2016). Spinous layer keratinocytes, similar to basal cells of the epidermis, are connected through desmosomes, Ca2+-activated cell cell junctions.<br><br>Keratinocytes of the spinosum layer differentiate into keratinocytes of the granulosum layer. Increased Ca2+ concentrations in the upper layer of epidermis induce transcription of granular cell markers, such as LORICRIN (Dlugosz and Yuspa 1993; Hohl et al. 1991) and FLG (filaggrin) (Dlugosz and Yuspa 1993) in a protein kinase C (PKC)-dependent manner. While Ca2+ also stimulates expression of KRT1 and KRT10, activation of PKC leads to rapid inhibition of KRT1 and KRT10 transcription and reduction of KRT1 mRNA stability. Activation of PKC inhibits EGF signaling. PKC-mediated inhibition of KRT1 and KRT10 transcription requires de novo mRNA and protein synthesis. Candidate PKC isoforms that stimulate differentiation of spinous keratinocytes into granular keratinocytes are four PKCs known to be expressed in cultured keratinocytes: PRKCA, PRKCD, PRKCZ, and PRKCH (Dlugosz and Yuspa 1993).<br><br>Besides Ca2+, another factor that stimulates differentiation of spinous into granular keratinocytes is hypoxia. Hypoxia leads to HIF1A and EPAS1 (HIF2A)-mediated stimulation of FLG transcription in a Ca2+ independent and cell confluency-independent manner. Hypoxia also stimulates expression of SPINK5, an inhibitor of epidermal serine proteases that regulates conversion of profilaggrin into monomeric filaggrin. Three putative hypoxia response elements (HREs) are found in the promoter region of the human FLG gene, two of which are functional. One of the two functional HREs is evolutionarily conserved. Direct binding of HIF1A/EPAS1 to these HREs has not been demonstrated. Simultaneous HIF1A and EPAS1 deficiency inhibits terminal differentiation of keratinocytes as well as formation of stratum granulosum and cornified envelope but does not affect MKI67 expression in basal layer keratinocytes or KRT10 expression in stratum spinosum (Wong et al. 2015). ARNT (Aryl hydrocarbon receptor nuclear translocator involved in ceramide synthesis) also positively regulates FLG expression without affecting expression of MKI67 in the basal layer or expression of KRT1 and KRT10 in the spinous layer of epidermis (Wong et al. 2015).<br><br>Retinoic acid (RA) inhibits transcription of LORICRIN and FLG in granular cells, even in the presence of high Ca2+ (Hohl et al. 1991; Magnaldo et al. 1992) but has little effect on IVL (involucrin) expression (Magnaldo et al. 1992). Critical cell density is also required for accumulation of LORICRIN mRNA (Hohl et al. 1991).<br><br>Inhibition of JNK signaling promotes differentiation of keratinocytes and induces the expression of known suprabasal adhesion proteins such as DSC1, DSC3, DSG1, and LY6D (Gazel et al. 2002). LY6D is specifically expressed in keratinocytes from the stratum spinosum at the RNA level, validated by FISH (Cheng et al. 2018) and by <i>in situ</i> sequencing (Ganier et al. 2024).<br><br>Markers of keratinocytes of the spinous layer are summarized in the "Table of markers of spinous keratinocytes in interfollicular epidermis". Some of the markers, in particular AQP3 and LGLAS7, are described in more detail in the section "Keratinocyte stem cell differentiates into transit amplifying cell in the basal layer of interfollicular epidermis".<br><br>FABP5, epidermal fatty acid binding protein also known as E-FABP, is a terminal differentiation marker expressed in upper layers of stratum spinosum and in stratum granulosum (Le et al. 1998). FABP5 is a lipid carrier, prevalently expressed in postmitotic keratinocytes of stratum spinosum and barely detectable in transit amplifying cells and basal cells; FABP5 may positively regulate KRT10 expression through NFKB and JNK signaling (Dallaglio et al. 2013).<br><br>Together with KRT10, KRT1 aggregates into thin bundles called tonofilaments (reviewed in Fuchs et al. 1990). IL4 and IL13 suppress expression of KRT1 and KRT10 in keratinocytes of stratum spinosum in atopic dermatitis (Totsuka et al. 2017).<br><br>KRT24 is expressed in upper layers of stratum spinosum and required for Ca2+ induced terminal differentiation of keratinocytes (Min et al. 2017). KRT24 is also expressed in upper layers of stratum spinosum in bioengineered skin (Klar et al. 2018).<br><br><br><table border ="1"><tr><b>Table of markers of spinous keratinocytes in interfollicular epidermis.</b> Please note that keratinocytes in CellMarker database and PanglaoDB correspond to spinous keratinocytes in Reactome.</tr><tr><th>Marker (protein/RNA)<th>Literature Reference<th>CellMarker database – RNA/Protein (Hu et al. 2022)<th>PanglaoDB – RNA (Franzén et al. 2019)</tr><tr><td>AQP3 (protein)<td>Sugiyama et al. 2001<br>Ma et al. 2002<br>Sougrat et al. 2002<td>No<td>Yes</tr><tr><td>FAPB5 (protein)<td>Le et al. 1998<br> Dallaglio et al. 2013<td>No<td>No</tr><tr><td>KRT1 (protein)<td>reviewed in Fuchs et al. 1990<br>Totsuka et al. 2017<td>No<td>Yes</tr><tr><td>KRT10 (protein)<td>Totsuka et al. 2017<td>No<td>Yes</tr><tr><td>KRT24 (protein)<td>Min et al. 2017<br>Klar et al. 2018<td>No<td>No</tr><tr><td>LGALS7 (protein)<td>Magnaldo et al. 1995<br>Umayahara et al. 2020<td>No<td>Yes</tr><tr><td>LY6D (RNA)<td>Gazel et al. 2003<br>Cheng et al. 2018<br>Ganier et al. 2024<td>No<td>No</tr></table>

毛囊间表皮的棘层角质形成细胞在棘层（棘层）中形成4-8层细胞结构（参见Fuchs 1990年综述）。尽管棘层角质形成细胞被认为是已进入分裂后阶段（参见Fuchs 1990年综述），但它们仍可对一些在传递扩增细胞中观察到的增殖标记物呈阳性，如MKI67和组蛋白H3在丝氨酸残基S11上的磷酸化。目前，Reactome中尚未注释通用的细胞增殖标记物。棘层角质形成细胞呈阳性表达有丝分裂标记物的百分比随年龄增长而降低，且在不同皮肤区域之间存在差异（例如，足底皮肤与躯干皮肤）（Nöske等人，2016年）。棘层角质形成细胞，类似于表皮的基底层细胞，通过桥粒连接，形成Ca2+激活的细胞间连接。<br><br>棘层角质形成细胞分化为颗粒层角质形成细胞。表皮上层Ca2+浓度的增加可诱导颗粒细胞标记物的转录，如LORICRIN（Dlugosz和Yuspa 1993年；Hohl等人，1991年）和FLG（filaggrin）（Dlugosz和Yuspa 1993年），这一过程依赖于蛋白激酶C（PKC）。Ca2+还刺激KRT1和KRT10的表达，但PKC的激活导致KRT1和KRT10转录的快速抑制和KRT1 mRNA稳定性的降低。PKC介导的EGF信号通路抑制需要新的mRNA和蛋白质合成。能够刺激棘层角质形成细胞分化为颗粒层角质形成细胞的候选PKC同源体是四种已知在培养的角质形成细胞中表达的PKCs：PRKCA、PRKCD、PRKCZ和PRKCH（Dlugosz和Yuspa 1993年）。<br><br>除了Ca2+之外，另一个刺激棘层细胞分化为颗粒层细胞的因素是缺氧。缺氧通过HIF1A和EPAS1（HIF2A）介导的方式，在Ca2+非依赖性和细胞汇合非依赖性的条件下刺激FLG的转录。缺氧还刺激SPINK5的表达，SPINK5是一种表皮丝氨酸蛋白酶抑制剂，调节原聚角蛋白向单体角蛋白的转化。人类FLG基因启动子区域中存在三个假定的缺氧反应元件（HREs），其中两个是功能性的。其中两个功能性HRE之一在进化上得到保守。HIF1A/EPAS1直接结合这些HREs的证据尚未发现。HIF1A和EPAS1的联合缺乏抑制角质形成细胞的终末分化以及棘层颗粒层和角质层的形成，但不会影响基底层角质形成细胞的MKI67表达或棘层中的KRT10表达（Wong等人，2015年）。ARNT（参与角鲨烯合成的芳香烃受体核转运蛋白）也正向调节FLG的表达，而不会影响基底层中MKI67的表达或表皮棘层中KRT1和KRT10的表达（Wong等人，2015年）。<br><br>维甲酸（RA）抑制颗粒细胞中LORICRIN和FLG的转录，即使在高Ca2+存在的情况下（Hohl等人，1991年；Magnaldo等人，1992年），但对IVL（involvedin）的表达影响很小（Magnaldo等人，1992年）。LORICRIN mRNA的积累也需要关键的细胞密度（Hohl等人，1991年）。<br><br>JNK信号通路的抑制促进角质形成细胞的分化，并诱导已知的上皮层粘附蛋白的表达，如DSC1、DSC3、DSG1和LY6D（Gazel等人，2002年）。LY6D在棘层角质形成细胞的RNA水平上特异性表达，由FISH（Cheng等人，2018年）和原位测序（Ganier等人，2024年）得到验证。<br><br>棘层角质形成细胞的标记物总结在“毛囊间表皮棘层角质形成细胞标记物表”中。其中一些标记物，特别是AQP3和LGALS7，在“角质形成干细胞在毛囊间表皮基底层分化为传递扩增细胞”一节中进行了更详细的描述。<br><br>FABP5，表皮脂肪酸结合蛋白，也称为E-FABP，是一种表达在上层棘层和颗粒层的终末分化标记物（Le等人，1998年）。FABP5是一种脂质载体，在棘层已进入分裂后阶段的角质形成细胞中广泛表达，在传递扩增细胞和基底层细胞中几乎不可检测；FABP5可能通过NFKB和JNK信号通路正向调节KRT10的表达（Dallaglio等人，2013年）。<br><br>与KRT10一起，KRT1聚集形成称为张力丝的细束（参见Fuchs等人，1990年综述）。IL4和IL13抑制特应性皮炎中棘层角质形成细胞中KRT1和KRT10的表达（Totsuka等人，2017年）。<br><br>KRT24在上层棘层表达，对于钙诱导的角质形成细胞的终末分化是必需的（Min等人，2017年）。KRT24也在生物工程皮肤的上层棘层表达（Klar等人，2018年）。<br><br><table border="1"><tr><b>毛囊间表皮棘层角质形成细胞标记物表。</b>请注意，CellMarker数据库和PanglaoDB中的角质形成细胞对应于Reactome中的棘层角质形成细胞。</tr><tr><th>标记物（蛋白/RNA）<th>文献引用<th>CellMarker数据库 – RNA/蛋白（Hu等人，2022年）<th>PanglaoDB – RNA（Franzén等人，2019年）</tr><tr><td>AQP3（蛋白）<td>Sugiyama等人，2001年<br>Ma等人，2002年<br>Sougrat等人，2002年<td>否<td>是</tr><tr><td>FAPB5（蛋白）<td>Le等人，1998年<br> Dallaglio等人，2013年<td>否<td>否</tr><tr><td>KRT1（蛋白）<td>参见Fuchs等人，1990年<br>Totsuka等人，2017年<td>否<td>是</tr><tr><td>KRT10（蛋白）<td>Totsuka等人，2017年<td>否<td>是</tr><tr><td>KRT24（蛋白）<td>Min等人，2017年<br>Klar等人，2018年<td>否<td>否</tr><tr><td>LGALS7（蛋白）<td>Magnaldo等人，1995年<br>Umayahara等人，2020年<td>否<td>是</tr><tr><td>LY6D（RNA）<td>Gazel等人，2003年<br>Cheng等人，2018年<br>Ganier等人，2024年<td>否<td>否</tr></table>