Ezh2 Is Essential for Patterning of Multiple Musculoskeletal Tissues but Dispensable for Tendon Differentiation.

An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting Ezh2 through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by Ezh2 for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of Ezh2 in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of Ezh2 signaling.

FGF signaling patterns cell fate at the interface between tendon and bone.

Tendon and bone are attached by a transitional connective tissue that is morphologically graded from tendinous to osseous and develops from bipotent progenitors that co-express scleraxis (Scx) and Sox9 (Scx+/Sox9+). Scx+/Sox9+ progenitors have the potential to differentiate into either tenocytes or chondrocytes, yet the developmental mechanism that spatially resolves their bipotency at the tendon-bone interface during embryogenesis remains unknown. Here, we demonstrate that development of Scx+/Sox9+ progenitors within the mammalian lower jaw requires FGF signaling. We find that loss of Fgfr2 in the mouse tendon-bone interface reduces Scx expression in Scx+/Sox9+ progenitors and induces their biased differentiation into Sox9+ chondrocytes. This expansion of Sox9+ chondrocytes, which is concomitant with decreased Notch2-Dll1 signaling, prevents formation of a mixed population of chondrocytes and tenocytes, and instead results in ectopic endochondral bone at tendon-bone attachment units. Our work shows that FGF signaling directs zonal patterning at the boundary between tendon and bone by regulating cell fate decisions through a mechanism that employs Notch signaling.

Differential effects of protein kinase C-eta on apoptosis versus senescence.

Protein kinase C-eta (PKCη) is considered an anti-apoptotic kinase, which promotes cell survival and chemoresistance in several cancers, including breast cancer. We have recently shown that PKCη positively regulates the anti-apoptotic protein Mcl-1 in breast cancer cells, and depletion of PKCη induced proteasomal degradation of Mcl-1. We therefore examined if depletion of PKCη would enhance cellular sensitivity to chemotherapeutic agents. Silencing of PKCη by siRNA attenuated apoptosis induced by doxorubicin and paclitaxel in both MCF-7 and T47D breast cancer cells. While silencing of Mcl-1 caused a substantial increase in apoptosis induced by doxorubicin, the combined knockdown of PKCη and Mcl-1 was less effective. Depletion of PKCη also caused an increase in the abundance of the cell cycle inhibitor p27 and a decrease in the clonogenic survival of MCF-7 and T47D cells. PKCη knockdown was associated with an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity but this increase was attenuated by knockdown of p27. The suppression of doxorubicin-induced apoptosis by PKCη knockdown was partially relieved when p27 was depleted. Since loss of proliferative capacity during senescence could cause resistance to chemotherapeutic drugs, our results suggest that PKCη knockdown inhibits apoptosis by inducing p27-mediated senescence.

Development of migrating tendon-bone attachments involves replacement of progenitor populations.

Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally, utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and the Gli1 lineage. Here, by performing a series of lineage tracing experiments in mice, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, whereas others remain stationary. Interestingly, in stationary entheses Sox9+ cells differentiate into the Gli1 lineage, but in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1+ progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

Protein kinase C-eta regulates Mcl-1 level via ERK1.

Protein kinase C (PKC)-eta (PKCη) is a member of the novel category of PKC family. It is overexpressed in breast cancer and was shown to inhibit apoptosis and contribute to chemoresistance. Since the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance, we investigated if PKCη regulates Mcl-1 level. Silencing of PKCη decreased Mcl-1 in several breast cancer cells, including MCF-7 and T47D cells. PKCη depletion had no effect on MCL1 mRNA but the decrease in Mcl-1 by PKCη knockdown was blocked by proteasomal inhibitors, such as MG132 and lactacystin. Moreover, knockdown of Mule (Mcl-1 ubiquitin ligase) prevented Mcl-1 downregulation caused by PKCη deficiency. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-ß, a substrate for Akt, had little effect on Mcl-1 downregulation caused by PKCη silencing. However, knockdown of PKCη but not PKCα, -δ or -ε caused a significant decrease in ERK (extracellular signal-regulated kinase) phosphorylation. Knockdown of ERK1 but not ERK2 decreased Mcl-1 level, and the decrease in Mcl-1 caused by PKCη knockdown was restored by ERK1 overexpression. These results suggest that PKCη utilizes the ERK signaling pathway to protect against ubiquitin-mediated proteasomal degradation of Mcl-1.

The unique protein kinase Cη: implications for breast cancer (review).

Deregulation of key signal transduction pathways that govern important cellular processes leads to cancer. The development of effective therapeutics for cancer warrants a comprehensive understanding of the signaling pathways that are deregulated in cancer. The protein kinase C (PKC) family has served as an attractive target for cancer therapy for decades owing to its crucial roles in several cellular processes. PKCη is a novel member of the PKC family that plays critical roles in various cellular processes such as growth, proliferation, differentiation and cell death. The regulation of PKCη appears to be unique compared to other PKC isozymes, and there are conflicting reports regarding its role in cancer. This review focuses on the unique aspects of PKCη in terms of its structure, regulation and subcellular distribution and speculates on how these features could account for its distinct functions. We have also discussed the functional implications of PKCη in cancer with particular emphasis on breast cancer.

Upregulation of PKCη by PKCε and PDK1 involves two distinct mechanisms and promotes breast cancer cell survival.

BACKGROUND: Protein kinase C (PKC) serves as the receptor for tumor-promoting phorbol esters, which are potent activators of conventional (c) and novel (n) PKCs. We recently showed that these activators induced selective upregulation of PKCη in breast cancer cells. The objective of this study is to understand unique regulation of PKCη and its importance in breast cancer. METHODS: The levels of PKC isozymes were monitored in breast cancer cells following treatment with inhibitors of kinases, proteasome and proteases by Western blotting. PKCε was introduced by adenoviral delivery. PKCη and PDK1 were depleted by siRNA silencing. Cell growth was determined by the MTT or clonal assay. RESULTS: The general PKC inhibitors Gö 6983 and bisindolylmaleimide but not cPKC inhibitor Gö 6976 led to substantial PKCη downregulation, which was partly rescued by the introduction of nPKCε. Inhibition of phosphoinositide-dependent kinase-1 (PDK1) by Ly294002 or knockdown of PDK1 also led to downregulation of basal PKCη but had no effect on PKC activator-induced upregulation of PKCη. Proteasome inhibitors blocked PKCη downregulation triggered by PDK1 inhibition/depletion but not by Gö 6983. PKCη level increased in malignant but not in non-tumorigenic or pre-malignant cells in the progressive MCF-10A series associated with activated PDK1, and knockdown of PKCη inhibited breast cancer cell growth and clonogenic survival. CONCLUSION: Upregulation of PKCη contributes to breast cancer cell growth and targeting either PKCε or PDK1 triggers PKCη downregulation but involves two distinct mechanisms. GENERAL SIGNIFICANCE: The status of PKCη may serve as a potential biomarker for breast cancer malignancy.

Novel regulation of protein kinase C-η.

Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, we have investigated the molecular mechanism of PKCη regulation. Several PKC activators, including phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate and indolactam V caused upregulation of PKCη, whereas the general PKC inhibitor Gö 6983, but not the conventional PKC inhibitor Gö 6976 led to the downregulation of PKCη. Upregulation of PKCη was associated with an increase in phosphorylation of PKCη. Silencing of phosphoinositide-dependent kinase-1, which phosphorylates PKCη at the activation loop, failed to prevent PKC activator-induced upregulation of PKCη. Knockdown of PKCε but not PKCα inhibited PKC activator-induced upregulation of PKCη. Thus, our results suggest that the regulation of PKCη is unique and PKCε is required for the PKC activator-induced upregulation of PKCη.

Two faces of protein kinase Cδ: the contrasting roles of PKCδ in cell survival and cell death.

Protein kinase Cδ (PKCδ) is a member of the PKC family that plays a critical role in the regulation of various cellular processes, including cell proliferation, cell death, and tumor promotion. Since the identification that PKCδ is a substrate for caspase-3, there has been overwhelming literature that linked PKCδ with proapoptotic signaling. While PKCδ generally functions as a proapoptotic protein during DNA damage-induced apoptosis, it can act as an antiapoptotic protein during receptor-initiated cell death. PKCδ has also been implicated in tumor suppression as well as survival of several cancers. The function of PKC-delta depends on various factors, including its localization, tyrosine phosphorylation, and the presence of other pro- and antiapoptoic signaling molecules. This review discusses the current literature on the contrasting roles of PKCδ in cell survival and cell death.