Frank Echtermeyer and Michael Streit contributed equally to this work.Received for publication June 12, 2000, and accepted in revised form December 8, 2000.The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with β 1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesionmediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN؊͞؊) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis.

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.The epidermis is a self-renewing stratified epithelium in which the loss of terminally differentiated cells from its surface is balanced by cells that leave the proliferative basal layer and enter differentiation (1, 2). Because a metabolically dead cornified cell envelope is the end point of epidermal differentiation, this process may be viewed as a specialized form of programmed cell death (3). Moreover, the apoptotic program and keratinocyte differentiation share overlapping signaling effector mechanisms (4). Notably, the caspase-3 cysteine protease, an integral component of the cell death machinery, was implicated recently in embryonic keratinocyte differentiation control downstream of Notch1 (5), and activation of the related caspase-14 has been reported during adult keratinocyte differentiation (6). Nevertheless, "canonical" apoptosis and epidermal differentiation are distinct processes, with diverse execution times and biological outcomes; the former leads to the elimination of individual dead cells from tissues within hours, whereas the latter relies on the survival and synchronized maturation of whole sheets of cells over the course of weeks. Thus, an outstanding question is how keratinocytes can activate arrays of death-inducing signals during differentiation and yet remain protected from premature death.A candidate pathway for the survival of differentiating keratinocytes is the signaling module formed by the Class IA PI3K 4 and the downstream serine-threonine kinase Akt effectors (Akt/PKB-1, -2, and -3 isoforms) (7). The PI3K family is divided into three distinct classes (Class I, II, and III) based on primary structure, substrate specificity, and mode of regulation (8). Class I PI3Ks include four distinct p110 catalytic isoforms, further divided into Class IA (-␣, -␤, -␦) and IB (-␥); among these, p110...

Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that co-operates with integrins during cellmatrix interactions for the assembly of focal adhesions and actin stress fibers and in the phosphorylation of focal adhesion kinase (FAK) on Tyr 397 . These cellular events are regulated by the small GTPase Rho, and in the absence of syndecan-4 ligation, cellular levels of GTP-bound Rho are decreased implicating syndecan-4 in the regulation of the small GTPases. In the present study we report that, compared with wild type cells, fibronectin-adherent syndecan-4-null fibroblasts showed enhanced lamellipodia and increased Rac1 activity that could be down-regulated by re-expression of syndecan-4 in the mutant cells. Consistent with the role for Rac1 in activating p38 and JNK signaling, syndecan-4-null cells display higher levels of active p38 MAPK and JNK that were abolished by the expression of a dominant-negative RacN17 mutant. Since p38 and JNK regulate gene expression by phosphorylating and activating transcription factors, we compared both the phosphorylation state and the transcriptional activity of the ATF-2 transcription factor, as a direct p38 and JNK target in syndecan-4-null and wild type cells. In the absence of syndecan-4, both ATF-2 phosphorylation and transcriptional activity were significantly more elevated compared with wild type cells, and both activities were decreased either by the re-expression of syndecan-4 or by the expression of RacN17. Our results reveal a novel function for syndecan-4 in modulating nuclear transcriptional activity and indicate an underlying mechanism that acts at the level of Rac1-p38/JNK signaling.The syndecans make up a family of four transmembrane heparan sulfate proteoglycans that, by virtue of their heparan sulfate side chains, can bind insoluble ligands such as extracellular matrix (ECM) 1 molecules and soluble ligands such as growth factors (1). Of all the syndecans, syndecan-4 is the most ubiquitously expressed. Of the remaining family members, syndecan-1 is primarily expressed in epithelia, syndecan-2 in connective tissue, and syndecan-3 in nervous tissues. A number of studies have shown that syndecan-4 is important in adapting to physiological stresses. Its expression is up-regulated in response to hypoxia (2) and to mechanical stress (3, 4). Loss of syndecan-4 in mice results in defective renal function (5), increased mortality due to septic shock (6), and in delayed wound healing due to a reduction in cell migration and impaired angiogenesis in the granulation tissue (7). A number of studies implicate syndecan-4 in the regulation of the small GTPase Rho in events associated with cell attachment and migration. First, syndecan-4 co-operates, in a Rhomediated manner, with integrins in the assembly of focal adhesions and actin stress fibers when cells adhere to the ECM molecule fibronectin (FN) (8). Focal adhesions are macromolecular complexes that are composed of transmembrane receptors and structural and signaling cytoplasmic molecules. Integrins and syndecan-4 ar...

How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.

Studies examining the role of the cell-surface heparan sulfate proteoglycan syndecan-4 have yielded a plethora of information regarding its role in both cell-matrix and growth-factor mediated signaling events. Many of the initial conclusions drawn from such research placed syndecan-4 in a keystone position within various signaling pathways though the generation of syndecan-4 null mice have surprised many in the field by indicating otherwise. These contradictory results place researchers in the frustrating and yet exhilarating position of still asking the question, "What role does syndecan-4 play in life?"

STAT3, a pleiotropic transcription factor acting downstream of cytokines and growth factors, is known to enhance proliferation, migration, invasion and aerobic glycolysis in tumors upon aberrant activation. In the murine epidermis, STAT3 is necessary for experimentally induced carcinogenesis. Skin tumorigenesis is conversely enhanced by overexpression in keratinocytes of the constitutively active STAT3C mutant, which also induces robust, psoriasis-like epidermal hyperplasia. We show here that STAT3C expression at physiological levels in knock-in mice leads to mild epidermal hyperplasia and attenuated expression of terminal differentiation markers. Altered differentiation is confirmed in isolated primary epidermal keratinocytes in vitro, correlating with enhanced proliferative and clonogenic potential, attenuated senescence and, strikingly, high-frequency spontaneous immortalization. These results suggest that moderate levels of continuous STAT3 activation, which closely resemble those triggered by chronic inflammation or persistent growth factor stimulation, may establish a preneoplastic state in part by promoting the escape of epidermal progenitor cells from differentiation and senescence checkpoints.