A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis.

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.

Enhancing Brain Retention of a KIF11 Inhibitor Significantly Improves its Efficacy in a Mouse Model of Glioblastoma.

Glioblastoma, the most lethal primary brain cancer, is extremely proliferative and invasive. Tumor cells at tumor/brain-interface often exist behind a functionally intact blood-brain barrier (BBB), and so are shielded from exposure to therapeutic drug concentrations. An ideal glioblastoma treatment needs to engage targets that drive proliferation as well as invasion, with brain penetrant therapies. One such target is the mitotic kinesin KIF11, which can be inhibited with ispinesib, a potent molecularly-targeted drug. Although, achieving durable brain exposures of ispinesib is critical for adequate tumor cell engagement during mitosis, when tumor cells are vulnerable, for efficacy. Our results demonstrate that the delivery of ispinesib is restricted by P-gp and Bcrp efflux at BBB. Thereby, ispinesib distribution is heterogeneous with concentrations substantially lower in invasive tumor rim (intact BBB) compared to glioblastoma core (disrupted BBB). We further find that elacridar-a P-gp and Bcrp inhibitor-improves brain accumulation of ispinesib, resulting in remarkably reduced tumor growth and extended survival in a rodent model of glioblastoma. Such observations show the benefits and feasibility of pairing a potentially ideal treatment with a compound that improves its brain accumulation, and supports use of this strategy in clinical exploration of cell cycle-targeting therapies in brain cancers.

TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching.

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation.

Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor-ß (TGF-ß) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-ß induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-ß-induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-ß stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.

The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma.

The proliferative and invasive nature of malignant cancers drives lethality. In glioblastoma, these two processes are presumed mutually exclusive and hence termed "go or grow." We identified a molecular target that shuttles between these disparate cellular processes-the molecular motor KIF11. Inhibition of KIF11 with a highly specific small-molecule inhibitor stopped the growth of the more treatment-resistant glioblastoma tumor-initiating cells (TICs, or cancer stem cells) as well as non-TICs and impeded tumor initiation and self-renewal of the TIC population. Targeting KIF11 also hit the other arm of the "go or grow" cell fate decision by reducing glioma cell invasion. Administration of a KIF11 inhibitor to mice bearing orthotopic glioblastoma prolonged their survival. In its role as a shared molecular regulator of cell growth and motility across intratumoral heterogeneity, KIF11 is a compelling therapeutic target for glioblastoma.

The SOX9 upstream region prone to chromosomal aberrations causing campomelic dysplasia contains multiple cartilage enhancers.

Two decades after the discovery that heterozygous mutations within and around SOX9 cause campomelic dysplasia, a generalized skeleton malformation syndrome, it is well established that SOX9 is a master transcription factor in chondrocytes. In contrast, the mechanisms whereby translocations in the --350/-50-kb region 5' of SOX9 cause severe disease and whereby SOX9 expression is specified in chondrocytes remain scarcely known. We here screen this upstream region and uncover multiple enhancers that activate Sox9-promoter transgenes in the SOX9 expression domain. Three of them are primarily active in chondrocytes. E250 (located at -250 kb) confines its activity to condensed prechondrocytes, E195 mainly targets proliferating chondrocytes, and E84 is potent in all differentiated chondrocytes. E84 and E195 synergize with E70, previously shown to be active in most Sox9-expressing somatic tissues, including cartilage. While SOX9 protein powerfully activates E70, it does not control E250. It requires its SOX5/SOX6 chondrogenic partners to robustly activate E195 and additional factors to activate E84. Altogether, these results indicate that SOX9 expression in chondrocytes relies on widely spread transcriptional modules whose synergistic and overlapping activities are driven by SOX9, SOX5/SOX6 and other factors. They help elucidate mechanisms underlying campomelic dysplasia and will likely help uncover other disease mechanisms.

AP1 transcription factors in epidermal differentiation and skin cancer.

AP1 (jun/fos) transcription factors (c-jun, junB, junD, c-fos, FosB, Fra-1, and Fra-2) are key regulators of epidermal keratinocyte survival and differentiation and important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because AP1 transcription factors regulate competing processes (i.e., proliferation, apoptosis, and differentiation). Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating AP1 transcription factors in epidermis. Taken together, these studies suggest that individual AP1 transcription factors have different functions in the epidermis and in cancer development and that altering AP1 transcription factor function in the basal versus suprabasal layers differentially influences the epidermal differentiation response and disease and cancer development.

Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells.

BACKGROUND: Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells. RESULTS: The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not. CONCLUSIONS: These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.

Identification of a central role for complement in osteoarthritis.

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of 'wear and tear'. Although low-grade inflammation is detected in osteoarthritis, its role is unclear. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in complement component 5 (C5), C6 or the complement regulatory protein CD59a, we show that complement, specifically, the membrane attack complex (MAC)-mediated arm of complement, is crucial to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints from C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Further, MAC colocalized with matrix metalloprotease 13 (MMP13) and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints has a key role in the pathogenesis of osteoarthritis.

Synergistic activation of human involucrin gene expression by Fra-1 and p300--evidence for the presence of a multiprotein complex.

Involucrin is expressed in the differentiated suprabasal epidermal layers, and an AP1 transcription factor-binding site present in the involucrin promoter distal regulatory region is required for this regulation. This site binds Fra-1, but cofactor interaction at this site has not been adequately characterized. We show that Fra-1 and p300 histone acetyltransferase are present at the AP1 site, as detected by chromatin immunoprecipitation. This interaction is functional, as treating p300 expressing keratinocytes with calcium or 12-O-tetradeconylphorbol-13-acetate, results in a synergistic increase in hINV expression, and this enhanced activation can be reproduced by coexpression of Fra-1 and p300. p300 also co-precipitates with Fra-1, but protein fractionation studies suggest that this interaction requires an additional protein. Fra-1 also interacts with other proteins that interact at the AP1-5 site, including JunD, JunB, Sp1, and P/CAF. Contrary to results in some other systems, Fra-1 functions as a positive transcriptional regulator in human keratinocytes. These studies suggest that a large multiprotein complex, which includes Fra-1, p300, P/CAF, junD, junB, and Sp1 acts at the AP1-5 site to produce a synergistic increase in hINV gene expression.

Opposing action of curcumin and green tea polyphenol in human keratinocytes.

Persistent environmental insult can convert a normal cell into a cancer cell. However, various natural chemopreventive agents called antioxidants can retard this progression. We have recently explored the effects of several chemopreventive agents, including green tea polyphenol and curcumin, on normal human keratinocyte function. Our findings suggest that a bioactive polyphenol from green tea, (-)-epigallocatechin-3-gallate (EGCG), acts to increase involucrin gene expression, suggesting that EGCG treatment enhances normal human keratinocyte differentiation. Mechanistic studies indicate that EGCG alters mitogen-activated protein kinase cascade function to activate involucrin gene transcription via a Ras, MEKK1, MEK3, ERK1/2-p38delta cascade that targets AP1 and CAATT enhancer binding protein transcription factors. These findings suggest that EGCG may inhibit disease progression by promoting keratinocyte differentiation. Parallel studies indicate that not all antioxidants produce a similar response. Curcumin, an antioxidant derived from the turmeric, antagonizes the EGCG-dependent response by interfering in this signaling pathway. These studies suggest that different antioxidant may produce antagonistic effects in tissues.

The distal and proximal regulatory regions of the involucrin gene promoter have distinct functions and are required for in vivo involucrin expression.

Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions - the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia.

h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.

Involucrin expression in the corneal epithelium: an essential role for Sp1 transcription factors.

PURPOSE: Identifying the mechanism(s) that regulate gene expression during the transition of the limbal stem cell to a differentiated superficial cell is an important area of interest in the corneal epithelium. METHODS: However, the factors that regulate gene expression during this process are not well understood. In the present study, the human involucrin (hINV) gene was used as a model to study gene expression in the corneal epithelium. Expression was studied in normal human corneal epithelial cell cultures and hINV promoter transgenic mice. RESULTS: Studies in cultured cells revealed that an Sp transcription factor-binding site, located in the upstream regulatory region of the hINV promoter, is essential for optimal hINV gene expression. Mutation of this site reduces promoter activity. Expression of Sp1 results in an Sp1-dependent increase in activity, whereas expression of dominant-negative Sp1 inhibits promoter activity. Gel mobility shift analysis showed the interaction of Sp1 and Sp3 with the Sp DNA element. Treatment of the corneal epithelial cells with 12-O-tetradecanoylphorbol-13-acetate increased hINV gene expression and this response is associated with increased nuclear factor binding of Sp1 and Sp3 to the Sp DNA response element. Promoter mutagenesis studies in transgenic mice confirmed the importance of the Sp site, as removal of this site by promoter truncation or point mutation resulted in a complete loss of in vivo corneal epithelial cell gene expression. CONCLUSIONS: These studies provide in vivo evidence that Sp transcription factor input is absolutely necessary for activation of involucrin gene expression in the differentiating corneal epithelium.

An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo.

PURPOSE: Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation. METHODS: Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells. RESULTS: Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element. CONCLUSIONS: These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.

Antioxidants regulate normal human keratinocyte differentiation.

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease progression. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive green tea antioxidant that possesses remarkable cancer chemopreventive properties. We have recently explored the hypothesis that EGCG prevents cancer by promoting keratinocyte differentiation. Based on our findings, we argue that EGCG acts to enhance the differentiation of normal keratinocytes. This is a potentially important finding, as it represents a novel mechanism of disease inhibition by EGCG--cancer preventive "differentiation therapy". However, not all antioxidant chemopreventive agents work by this mechanism. Curcumin, for example, inhibits the differentiation-promoting activity of EGCG. This report discusses the mechanism of EGCG and curcumin action in regulating expression of involucrin, a marker of keratinocyte differentiation.

Regulation of involucrin gene expression.

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

p38 Mitogen-activated protein kinases on the body surface--a function for p38 delta.

The p38 family of mitogen-activated protein kinases includes p38 alpha (SAPK2a, CSBP), p38 beta (SAPK2b), p38 delta (SAPK4), and p38 gamma (SAPK3/ERK6). p38 alpha and p38 beta are widely expressed p38 isoforms that are involved in regulation of cell proliferation, differentiation, development, and response to stress. Relatively less is known regarding the function of the p38 delta isoform. In this review, we discuss the role of the p38 alpha, p38 beta, and p38 gamma isoforms and then present recent findings that define a role for p38 delta as a regulator of differentiation-dependent gene expression in keratinocytes.