Concise Review: Reprogramming, Behind the Scenes: Noncanonical Neural Stem Cell Signaling Pathways Reveal New, Unseen Regulators of Tissue Plasticity With Therapeutic Implications

Abstract

Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to β-like cells). Specifically for acinar-to-β-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research.

Significance: Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations.

Keywords: Cellular reprogramming; Cellular transdifferentiation; Hes3 protein; Induced pluripotent stem cells; Pancreatic islets; STAT3 transcription factor; Signal transduction.

Figure 1.

Noncanonical signaling pathway regulation during reprogramming. (A): Extracellular factors lead to the phosphorylation of STAT3-Tyr via JAK activation or STAT3-Ser via MAPK, Akt, and mTOR activation, and subsequent Hes3 transcription. The two pathways are opposing (e.g., JAK activity in neural stem cells [NSCs] suppresses induction of Hes3). Some cell types (e.g., primary NSCs) are confined to using the STAT3-Ser branch, because the STAT3-Tyr branch leads to their irreversible differentiation. Other cell types (e.g., primary cancer stem cells from glioblastoma multiforme patients and MIN6 cells) grow effectively using either pathway and, through repeated changes in cell culture conditions, can switch their signaling state back and forth. (B): Genes in the STAT3-Ser/Hes3 signaling axis are regulated during mouse fibroblast reprogramming. Sox21, Hes3, and Shh gene expression increases as MEFs transition to SSEA1+ and then to Oct4+ populations during reprogramming to the pluripotent state. Hes3 and Shh are downregulated in resultant stable mouse iPS cells grown in culture conditions that activate JAK (lines not to scale; expression levels at the MEF stage normalized to help visualize patterns and trends). (C): Genes in the STAT3-Ser/Hes3 signaling axis are regulated during neural specification of hES cells. The diagram summarizes the expression patterns of Hes3, Bmi1, and JAK1 over the course of a 77-day protocol to differentiate the human ES cell line WA09 to dorsal telencephalic neuronal fates (lines not to scale; expression levels at day 0 of ES cell stage normalized to help visualize patterns and trends). (B, C): The concepts shown are from gene expression data previously published and reanalyzed for the purposes of the present report [25]. Abbreviations: CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; hES, human embryonic stem (cell); Hes3, hairy and enhancer of split 3; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblasts; mIPS, mouse induced pluripotent stem (cell); SSEA1, stage-specific embryonic antigen 1; Shh, sonic hedgehog; STAT3, signal transducer and activator of transcription 3.

Figure 2.

Possible noncanonical signaling pathway involvement in reprogramming through modulators of STAT3 and MAPK. (A): Common view for the mechanism of reprogramming in acinar-to-β-cell reprogramming downstream of CNTF and EGF. STAT3-Tyr and activated MAPK induce vast transcriptional changes leading to fate specification changes. (B): Hes3 as a regulator of Ngn3 in the context of endocrine pancreas regeneration. A lack of Ngn3 expression induction in Hes3-null (Hes3−/−) mice 5 months after a low-dose streptozotocin regimen (5 consecutive daily injections at 50 mg/kg in phosphate-buffered saline [PBS]; vehicle controls received only PBS). (C): STAT3-Ser as a putative mediator of reprogramming of acinar-to-β cell conversion. Three possible alternative interpretations for the mechanism of action of CNTF- and EGF-induced reprogramming that involve the STAT3-Ser/Hes3 signaling axis. (Both CNTF and EGF lead to the phosphorylation of STAT3-Ser and STAT3-Tyr; the diagrams highlight the particular phosphorylation event that might be driving a given function. It is not meant to suggest that only one residue is phosphorylated. Also, a predominant function of STAT3-Tyr phosphorylation is the dimerization of STAT3. For this reason, and for simplicity, the diagrams depict STAT3-Tyr phosphorylation to also represent STAT3 dimerization). (B): Image width: 534 μm. Abbreviations: CNTF, ciliary neurotrophic factor; DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor; Hes3, hairy and enhancer of split 3; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; STAT3, signal transducer and activator of transcription 3; STZ, streptozotocin.