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Normal human epidermal keratinocytes (NHEK) grown in serum-free medium on a plastic substrate spontaneously differentiate at high cell densities in vitro. Because protein kinase C (PKC) regulates murine keratinocyte differentiation triggered by a variety of stimuli, we examined the role of this signaling pathway in density-dependent activation of NHEK differentiation. Relative to subconfluent cultures, confluent NHEK expressed markedly higher levels of multiple differentiation markers assayed by immunoblotting, including keratin 1, loricrin, filaggrin, involucrin, TGK, and SPR-1. Expression of several of these markers continued to increase for several days after cells reached confluency. The total level of several PKC isoforms was not substantially altered in NHEK harvested at different cell densities, based on immunoblotting; however, subcellular fractionation revealed that PKCalpha underwent a redistribution to the particulate fraction in confluent and postconfluent NHEK cultures, suggesting that this isozyme was activated under these conditions and may be involved in triggering the terminal differentiation program. Supporting this concept, inhibition of PKC function using bryostatin 1 or GF 109203X blocked the induction of keratinocyte differentiation markers at high cell densities. These data suggest that endogenous activation of PKC is responsible for cell density-mediated stimulation of NHEK differentiation, establishing a critical role for this pathway in regulating human as well as murine keratinocyte differentiation.

Growth and differentiation factor 11 (GDF11) and myostatin (MSTN) are closely related transforming growth factor β (TGF-β) family members, but their biological functions are quite distinct. While MSTN has been widely shown to inhibit muscle growth, GDF11 regulates skeletal patterning and organ development during embryogenesis. Postnatal functions of GDF11, however, remain less clear and controversial. Due to the perinatal lethality ofGdf11null mice, previous studies used recombinant GDF11 protein to prove its postnatal function. However, recombinant GDF11 and MSTN proteins share nearly identical biochemical properties, and most GDF11-binding molecules have also been shown to bind MSTN, generating the possibility that the effects mediated by recombinant GDF11 protein actually reproduce the endogenous functions of MSTN. To clarify the endogenous functions of GDF11, here, we focus on genetic studies and show thatGdf11null mice, despite significantly down-regulatingMstnexpression, exhibit reduced bone mass through impaired osteoblast (OB) and chondrocyte (CH) maturations and increased osteoclastogenesis, while the opposite is observed inMstnnull mice that display enhanced bone mass. Mechanistically,Mstndeletion up-regulatesGdf11expression, which activates bone morphogenetic protein (BMP) signaling pathway to enhance osteogenesis. Also, mice overexpressing follistatin (FST), a MSTN/GDF11 inhibitor, exhibit increased muscle mass accompanied by bone fractures, unlikeMstnnull mice that display increased muscle mass without fractures, indicating that inhibition of GDF11 impairs bone strength. Together, our findings suggest that GDF11 promotes osteogenesis in contrast to MSTN, and these opposing roles of GDF11 and MSTN must be considered to avoid the detrimental effect of GDF11 inhibition when developing MSTN/GDF11 inhibitors for therapeutic purposes.

Acetaminophen is widely used for pain management as an alternative to NSAIDs and selective COX-2 inhibitors, but its action at a molecular level is still unclear. We evaluated acetaminophen's effect on PG release and the expression patterns of genes related to PG production in a clinical model of tissue injury and acute inflammation. Subjects (119 outpatients) received either 1000 mg acetaminophen, 50 mg rofecoxib (a selective COX-2 inhibitor), 30 mg ketorolac (a dual COX-1/COX-2 inhibitor), or placebo before the surgical removal of two impacted mandibular third molars. Microdialysis was used to collect inflammatory transudate from the surgical site for measurement of PGE2 and TXB2 levels at the site of injury. Biopsies were collected to investigate the expression patterns of genes related to PG production at baseline prior to surgery and at 3 or 24 h following surgery. PGE2 release was suppressed by ketorolac, rofecoxib and acetaminophen compared to placebo at 3 h coincident with increased COX-2 gene expression in biopsies collected from the surgical site. TXB2 release was suppressed only by ketorolac. COX-2 gene expression remained elevated at 24 h with continued ketorolac and acetaminophen treatment. COX-1 gene expression was significantly down-regulated at 24 h by ketorolac, rofecoxib and acetaminophen. Acetaminophen suppression of PGE2 without inhibiting TXB2 release, when COX-2 gene expression is up-regulated, suggests that acetaminophen is a selective COX-2 inhibitor in vivo. The up-regulation of COX-2 gene and down-regulation of COX-1 gene expression suggests that acetaminophen may result in changes in COX-derived prostanoids with repeated doses.

Heat shock protein 27 (HSP27), induced by heat shock, environmental, and pathophysiological stressors, is a multi-functional protein that acts as a protein chaperone and an antioxidant. HSP27 plays a significant role in the inhibition of apoptosis and actin cytoskeletal remodeling. HSP27 is upregulated in many cancers and is associated with a poor prognosis, as well as treatment resistance, whereby cells are protected from therapeutic agents that normally induce apoptosis. This review highlights the most recent findings and role of HSP27 in cancer, as well as the strategies for using HSP27 inhibitors for therapeutic purposes.

These results suggest that wide variability in gene expression and functional polymorphisms in PTGS2 may explain part of the interindividual variations in acute pain and the analgesic efficacy of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors; this may be useful to define individual responders on the basis of genetic variations to predict patient risk and benefit to drugs.